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Ketosis in infants and children
MEDICAL PROGRESS
Ketosis in infants and children
j. B. Sidbury, Jr., M.D., * and Boek L. Dong, M.D. BALTIMORE,, MD.
albumin,2, a are then transported to the tissues of the body for metabolism. Transported fatty acids are utilized by many of the cells of the body as a metabolic fuel; the brain is a notable exception. * Acetoacetate is one of the products of fatty acid oxidation. Acetone and fl hydroxybutyrate are formed from acetoacetate. 4 Although acetoacetate formation has been demonstrated in the lactating mammary gland, 5 kidney, ~ and muscle7 blood ketone bodies are derived from the liver. It should be noted that ketone bodies are present in the blood of normal persons and that ketosis represents an accumulation of normal metabCites. The fatty acids which are transported from adipose tissue via serum albumin are taken up by the liver and may either be synthesized into triglycerides or may be sequentially oxidized, 2 carbon fragments at a time as originally suggested by Knoop. s This is effected by activation of the fatty acid with acetyl eoenzyme A. The oxidation of fatty acids occurs exclusively in the mitochondria of the cell. The enzymatic path-
K E T O S I S is a condition which is frequently encountered by those treating children. It is a manifestation of altered metabolism and is almost invariably associated with some other disease process. Ketosis is frequently associated with diabetes, fasting, diarrhea, vomiting, and glycogen storage disease. Some patients are quite prone to develop ketosis and do so with any febrile illness. The particular interest of the pediatrician is based on the greater susceptibility of children and on the intriguing paradox that young infants are more resistant to ketosis than either the child or the adult. The first step in the elaboration of ketone bodies occurs in the adipose tissue, where a lipase hydrolyzes the individual fatty acids from the depot form of fat, triglycerides?, 2 The nonesterified fatty acids, bound to serum From the Department of Pediatrics, The Johns Hopkins University School o[ Medicine and the Johns Hopkins Hospital. This work was supported in part by Grant B-1626 from The National Institutes of Health, Department of Health, Education and Welfare. *Presen~ Address, Department o[ Pediatrics, Duke University School o[ Medicine, Durham, N. C.
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ways for oxidation and synthesis are distinct.k, s We have indicated that the ketone bodies found in the blood arise principally from the liver. Acetoacetate is the ketone body first produced by the liver, and the other 2 are derived from it 9 (Fig. 1). The explanation of the fact that the liver is the site of origin of the ketone bodies of blood lies in the difference in the pattern of enzymes in the liver and peripheral tissues (vide infra). The following reactions are pertinent. ~ Acetoacetyl-CoA synthesizing reactions: Thiolase >
1. 2AcCoA < AcAcCoA + CoA Aeetoacetate activating enzyme > 2. AcAc + CoA + ATP < AcAcCoA + AMP + PP (See also reaction 5.) Formation of acetoacetate: Deacylase 3. AcAcCoA + H20 > AcAc + CoA HMG-CoA condensing enzyme >
4. AcAcCoA + AcCoA < 3 hydroxy3-methylglutaryl-CoA Cleavage enzyme HMG-CoA > AcAc + AcCoA CoA transferase >
5. AcAcCoA + Succinate < Succinyl CoA + AcAe Aeetoacetyl glutathione thioesterase >
6. AcAcCoA + GSH < CoA AcAc -SG + H20
AcAc -SG + > AcAc + GSH
~Abbreviatlons used: AcAc, acetoacetate; AcCoA, acetyl coenzyme A; AcAcCoA, acetoacetyl eoenzyme A; /3Oit, flhydroxybutyrate; AMP, adenosine monophosphate; ADP, adenosine dlphosphate; ATP, adenosine triphosphate; DPN, DPNH, dlphosphopyrldine nueleotide, oxidized and reduced; TPN, TPNH, triphosphopyridine nudeotlde, oxidized and reduced; GSH, reduced glutathlone; HMP, hexose monophosphate shunt; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CoA, coenzyme A; UFA, unesterified fatty adds; ACTH, adrenocorticotrophle hormone; TSH, thyroid stimulating hormone.
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There is no agreement on whether acetoacetate is formed mainly by reaction 3 or 4. There is no conclusive evidence to support one theory rather than the other. Recently, a study of ketone body formation in the tissues from the rumen and omasum of the sheep with the use of C ~4 butyrate suggests that the acetoacetate formed was derived by HMP-CoA. 1~ For the present, a final judgment should be withheld until it can be determined to what extent in vitro conditions determine the resultant labeling and hence the conclusions. Leucine has been shown to be converted to acetate by way of acetoacetate? ~ Reactions 2 and 5 do not occur in the liver. The other reactions are present in the liver and in the peripheral tissues. Reaction 6 is postulated--not p r o v e d - - b u t it could occur nonenzymatically. TM Since reactions 2 and 5 are absent in the liver, acetoacetate, once formed, must pass into the circulating blood to be metabolized by the peripheral tissues. A certain portion is converted into fiOH before it leaves the liver: fl hydroxybutyrate dehydrogenase 7. AcAc + DPNH < flOH + DPN Thus the liver possesses the enzymes necessary for forming acetoacetate and flOH but lacks an enzyme necessary for their utilization. The peripheral tissues have the enzymes necessary for their utilization. The second phase of ketone metabolism to consider is utilization. It is apparent from the above discussion that, once formed in the liver, ketone bodies are utilized b y other tissues. It is assumed that flOH is used by the tissues through the reverse reaction of flOH dehydrogenase and then esterification of the acetoacetate with coenzyme A via the thiolase or the coenzyme A transferase reactions. 13 HORMONAL KETOSIS
EFFECTS
ON
A particular hormone may demonstrate an effect in adipose tissue which is conducive to ketosis but the effect induced in the liver
2 9 6 Sidbury and Dong
is antiketogenic (e.g., cortisol); therefore, the effect of a hormone in the intact animal and in isolated tissues in vitro may be opposite (Table I). Virtually all of the hormones affect adipose tissue either directly or indirectly? It will be immediately apparent that a given hormone may either have no effect on adipose tissue, may stimulate glucose uptake, and inhibit fatty acid release, or it may promote the release of fatty acids. Cortisone and cortisol suppress the ketosis associated with cold stress and fluoroacetate (a Krebs cycle poison), but they have no effect on ketosis due to insulin hypoglycemia or administration of pituitary extract? 4 The antiketogenic effect of glucocorticoids is due in part to the fact that they stimulate fat synthesis and gluc0neogenesis. Cortisol effects a release of fatty acids from adipose tissue in vitro2 5 Epinephrine, norepinephrine, ACTH, growth hormone, and glucagon cause in vitro and in vivo fatty acid mobilization under certain conditions and an increase in ketone body production in vivo. 1, 18 Epinephrine has been shown to activate phosphorylase by stimulating the synthesis of 3', 5' adenylic acid9 7 and recently the latter has been shown to have a direct ketogenic elfeet? s The association of insulin deficiency and ketosis is the hormonal relation that stands out in the minds of everyone. Not only has ketosis long been established as a feature of diabetes, but it is recognized as a sign of poor control--a warning sign. A great deal of study has been undertaken to determine the relation of insulin to excess ketonemia. A deficiency of insulin in the diabetic animal and man has been shown to be associated with increased serum unesterified fatty acid levels, increased acetoacetate formation from fatty acids, and markedly diminished fatty acid synthesis. Simultaneously, the deprivation of insulin results in an elevation of blood glucose, an increased rate of hepatic gluconeogenesis, decreased glucose utilization, decreased rate of incorporation of amino acids into protein, and increased total body oxygen consumption. The administration of in-
February 1962
snlin is associated with a decrease in serum unesterified fatty acids, decrease in acetoacetate formation, and resumption of normal fatty acid synthesis. It suppresses acetoacetate synthesis from fatty acids more than the oxidation to carbon dioxide. The effect of insulin is not apparent on short-chain fatty acids? 9 On t h e other hand insulin in excess induces ketosis. The mechanism envisaged in this situation is that homeostatic mechanisms are brought to bear which, broadly speaking, are directed toward returning the blood sugar to normal; and as a corollary glucose utilization is reduced to a minimum, and other substrates are used either as an energy source or as a carbohydrate source. Thus, proteins (amino acids) are converted to glucose, and fatty acids are mobilized at an accelerated rate. It is postulated that epinephrine is released during hypoglycemia and may he important in these reactions? 4 Thus, the hormones which favor the development of ketosis uniformly stimulate fatty acid mobilization from the fat depots, suppress the respiratory quotient, reduce glucose utilization, and frequently inhibit insulin action. METABOLIC IN KETOSIS
INTERRELATIONS
A very important consideration, necessary to the understanding and evaluation of ketosis in any particular situation, is the relationship of fatty acid oxidation and synthesis to the Krebs cycle and to glucose metabolism. We have attempted to illustrate these interrelations in Fig. 2. It has been emphasized that fatty acid oxidation results in the formation of one acetyl coenzyme A molecule for each pair of carbon atoms of the fatty acid. As indicated in Fig. 2, acetyl coenzyme A has a number of routes open to it. It may condense with oxaloacetate to form citrate and be oxidized to COs and water through the cycle; this is probably the major pathway. The oxidation of acetate via the Krebs cycle is an energy generating sequence of events. On the other hand, acetyl coenzyme
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Ketoszs
Table I. Effect of hormones on adipose tissue r Promote glucose uptake and inhibit fatty acid release Insulin Prolacfin
Stimulate fatty acid release Epinephrine Norepinephrine ACTH TSH Growth hormone Glucagon Lipid mobilizing factors (Chalmers, 5o Rudman, 5v Seifter 52) Cortisol
~'After E,ngel.1
A may form acetoacetyl coenzyme A which can proceed to cholesterol synthesis, fatty acid synthesis, or ketone body formation. The synthesis of both fatty acids and cholesterol requires the reduced nucleotide, T P N H . T h e factors regulating the direction in which acetoacetate coenzyme A proceeds are not clear. Acceleration of acetoacetate to fl hydroxybutyrate by certain substrates has been explained by increased supply of reduced DPN. 4 iIt has been shown that, under the circumstances, the rate of oxidation in the mitochondria may be limited by the process of phosphorylation. 2~ The latter requires an adequate and available supply of ADP. Utilization of glucose involves phosphorylation by ATP, and ADP is a product. Thus the regeneration of ADP for oxidative phosphorylation may be one way in which glucose "spares" fat oxidation. Another potential role of glucose in fat metabolism is the generation of T P N H by the oxidation of glucose6-phosphate through the hexose monophosphate shunt. TPNI-t has been shown to be necessary for cholesterol and fatty acid synthesis. Glucose also spares fatty acid oxidation by decreasing the release of fatty acids from adipose tissues 21 and increasing incorporation of fatty acids into glycerides.22 OVERPRODUCTION VERSUS UNDERUTILIZATION
It would seem apparent that increased levels of ketone bodies in the blood and
29 7
urine would of necessity have to result from either an increased production of these products or their parent metabolites or a decreased rate of their catabolism. All evidence points to a markedly increased rate of mobilization and production of fatty acids in those conditions associated with ketosis. Thus overproduction of the metabolites which are the source of ketone bodies is present. The question remaining is whether under-utilization in any way contributes to ketosis. This proposition can be divided into three phases of inquiry. Whether or not there is a decreased level of oxaloacetate and hence the rate of oxidation of fatty acids is secondarily decreased is an old hypothesis. The evidence at hand indicates that there is no decrease in oxaloacetate either in diabetic or fasting ketosis. TM 24 Furthermore, it is clear that there is in fact an increased rate of oxidation of fatty acids in those conditions associated with ketosis. 25 The next question asked was if Krebs cycle intermediates are added, would the rate of oxidation of fatty acids via the Krebs cycle increase and the formation of acetoacetate decrease? With the proper in vitro conditions Krebs cycle intermediates can be shown to decrease acetoacetate formation. 2~ In addition Krebs cycle inhibitors both in vivo and in vitro .result in the accumulation of acetoacetate?4, 2,. 28 The response to the administration of Krebs cycle intermediates to an animal with ketosis has given rise to conflicting reports. A fundamental consideration here is whether under the conditions conducive to ketosis the Krebs cycle is operating at a maximal rate. In any series of reactions one encounters a slow step, relative to the rates of the other reactions. It is not possible to answer this question at the present time. It is clear that K the rate of oxidation of fatty acids by way of the Krebs cycle could be increased there would be a lessened production of acetoacetate. The second consideration in underutilization is the rate of peripheral utilization. One must assume that the rate of utilization of ketone bodies by the peripheral tissues is proceeding at a near maximal rate for the
2 98
Sidbury and Dong
OPNH AcCoA ~
AcAcCoA
.
DPN
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BOHCoA ,r
~.
CaOTONYLCoA
"~ F.A.
DPId89
LEUCINE ~
BOH
H MG CoA ~
AC AC ~
CHOLESTEROL
TYROSINE ACETONE
1 pHENYLALANINE
Fig, 1, This schema illustrates the pathways of origin of acetoacetate from acetyl CoA (AcGoA), leucine, and phenylalanine. T h e derivation of the other 2 ketone bodies, acetone and fl hydroxybutyrate ( # O H ) , from acetoacetate is indicated. The reaction in fatty acid synthesis and cholesterol synthesis in mitochondria requiring the reduced nucleotide T P N H is noted.
G;ycogen
I GlucoseI - P ~ ATP ADP
II
UDPG
UTP
PP
Fructose6-P
~OH
T~ Fruciose 1,6-P 11
t~
Tr,oso,
HMG-CoA -
2 DPNH + 2ATP-~-"ii coz + ADP P- Pyruvote~
/ II ~e",/
II
- I " ~ . . J ' ' X
Amino ocid~DPNH~r~[; Pyruvote + CO;:TPNH~TPN M ~ e
OPN"~"]}" Lgclote
Eotfy acids
" Ac-Ac-CoA |
TP,.,,. ~I It
) Acetylotion .... ~Amino acids
Y/)~I-TPN'~,-TPNH
Su~cc ~L Ketoglufarofe inYI- CoA~/~LCOz
Fig. 2. This metabolic map illustrates some of the known and possible interrelationships relating to acetyl-CoA metabolism. T h e reactions involving nucleotide coenzymes are indicated. T h e reactions of the Krebs cycle and those immediate to AcCoA are catalyzed in or on the mitochondria; the other reactions (glycolysis and the H M P shunt) occur in the nonparticulate portion of the cell.
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substrate level remains high. It is possible then that the accumulation in the blood is due to a reduced rate of utilization by the peripheral tissues. The evidence is to the contrary. The peripheral tissues are utilizing acetoacetate and flOH at a greatly increased rate during ketosis. 29 The third consideration in underutilization is regarded under this heading with misgivings. The question of metabolic regulators, or naively state metabolic compounds which direct traffic, is pertinent but has received little attention. Here we have in mind substances which would act to increase the formation of acetoacetate, though the regulator is not a direct participant or substrate in the reaction. Too little attention has been given this consideration to adduce a compelling case for it. ALTERATION LEVELS
IN ENZYME
It has recently been demonstrated that the activity of one or more of the enzyme systems concerned with formation of aeetoacetate from acetoacetate coenzyme A is increased in the liver of the diabetic rat2 ~ It is proposed that this may be the mechanism for ketosis in diabetes. However, since it has been shown that infusion of oetonoate results in a marked increase in ketone body formation in the normal individual, 14 this would suggest that the enzyme systems concerned with the reaction of acetoacetyl coenzyme A to acetoacetate are not the rate limiting steps. It appears more likely that the observed increase in enzyme activity is an effect rather than a cause, similar to the increase in glucose-6-phosphatase activity which has also been observed in the diabetic rat. 19 Glock and McLean 31 have found a decreased level of glucose-6-phosphate dehydrogenase in the liver of diabetic rats. The importance of this finding relates to the question of the role of T P N H generated by the hexose monophosphate shunt, as opposed to that generated by the isocitric dehydrogenase reaction, in the regulation of fatty acid synthesis. Recent work supports the
Ketosis
299
primary role of the H M P shunt in fatty acid synthesisY 2 On the other hand, another laboratory has reported the surprising finding that the hydrogen in position 2 of lactate was more efficiently incorporated into synthesized fatty acids in lactating mammary gland slices than was hydrogen from position 1 of glucose. The latter would be donated to T P N H by way of the H M P shunt. A mechanism is postulated. 33 PRECIS
Ketosis, then, results when fatty acids are mobilized from the adipose depots and presented to the liver for metabolism at a rate greater than the capacity of the liver to oxidize them. The overflow, as it were, goes into formation of acetoaeetate which in turn gives rise to acetone and fl hydroxybutyrate. The condition which generally prevails when ketosis arises is that of tissue carbohydrate deprivation. In fasting there is a deprivation of the intake of carbohydrate; in diabetes the carbohydrate bathing the cells cannot gain entrance to the cell. A number of hormones stimulate fatty acid release from adipose depots. Insulin, which inhibits fatty acid mobilization, is decreased in diabetes and probably in fasting. The precise manner in which glucose affects the production of aeetoacetate, and the nature and role of various regulators of acetoacetate formation are not clearly delineated. DIABETES
For our purposes diabetes can be simply described as a condition in which there are metabolic aberrations secondary to a deficiency of insulin relative to the cellular requirements for normal metabolism. The cells are deprived of glucose due to an interference of glucose transport. Since glu. cose metabolism is severely depressed, the cells must of necessity utilize fatty acids as the source of energy under these circumstances. It has indeed been found that the level of serum fatty acids is markedly elevated in the diabetes of the untreated indi-
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February 1962
vidual or the diabetic in ketosisY Fatty acids are mobilized at a rate in excess of the ability of the liver to oxidize them and the excess is diverted into ketone body formation. This is not totally wasteful for the peripheral tissues utilize the major proportion of the ketone bodies formed as an energy source, and only a small fraction is excreted into the urine. The amount in the urine is significant quantitatively; therefore, the over-all process is quite inefficient. It is of interest that ketosis is not as universally associated with diabetes in the infant as it is in the juvenile patient with diabetesY ~ Ketosis in the patient with diabetes.indicates the degree of insulin deprivation and the extent to which the metabolism has been altered. FASTING
KETOSIS
It would seem reasonable, in the light of the experimental evidence available, to conelude that basically the mechanisms operative in the production of ketosis are similar in ketosis due to fasting. The difference would be one of degree and the particular. This unified view results from ignorance in part, and the similarities may be found to be more apparent than real when the factors involved in regulation are appreciated. There seems little doubt that there is an increased mobilization of fatty acids which exceeds the capability of oxidation by the liver. OBESITY
It has been observed that obese adults are markedly less susceptible to fasting ketosis than are normal or thin adults2 ~ The very occasional observation in obese children suggests that this finding is peculiar to adults. GLYCOGEN
STORAGE
DISEASE
Certain forms of glycogen storage disease are also associated with ketosis. The types which are associated with ketosis are those associated with an absence of glucose-6phosphatase, decreased amylo-l,6-glucosidase (limit dextrinosis), and those associated with decreased phosphorylase activity in the
liver. It is of interest that those persons who tend to be more severely ketotic have a marked decrease in ketonemia in a postabsorptive state, but the level is rarely found to be normal in spite of a normal blood sugar. It is of particular interest that patients with glucose-6-phosphatase deficiency represent one of the few conditions that manifest ketosis in the neonatal period. In the first and third types of glycogenosis mentioned there is no reason to believe that muscle metabolism should be altered; hence, it seems unlikely that decreased utilization of ketone bodies by muscle plays a significant role in ketonemia exhibited by these patients. It seems pertinent that in patients with a glucose-6-phosphatase defect there is a large excess of mobilizable glycogen, but ketosis is a prominent feature of the clinical syndrome. The liver could hardly be considered deficient of glucose-6-phosphate. This observation increases the enigma of the mechanism of the ketolytic effect of glucose, suggesting perhaps that the suppressive effect of glucose on fatty acid release from adipose depots is the more important of its postulated roles. KETOSIS ASSOCIATED WITH VOMITING AND DIARRHEA
In the practice of pediatrics, one encounters ketosis most frequently in association with vomiting and diarrhea. The mechanisms involved have not been studied. The association is seen most dramatically in the condition described as recurrent or cyclic vomiting. Some of these patients have a marked hypoglycemia; therefore, the mechanism invoked for fasting ketosis would be applicable? 6 In addition, the general nutrition of these patients is usually very poor, and it would be reasonable to suppose that their liver glycogen supply is below normal and the protein available for gluconeogenesis is minimal; this explains the rapidity with which they go into ketosis. A relation between the degree of ketosis and a reduction in liver glycogen has been shown, but it is not primary to the induction of ketosis. ~7, 3~ Some patients with cyclic vomiting have a
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Ketosis
normal blood sugar level, and others have been described with a high level? 9 The latter might be considered to be associated with a transient or nutritionally induced hypoinsulinism. The primary metabolic alteration which effects decreased glucose utilization in patients with vomiting and diarrhea is not clear. One could postulate that cellular dehydration interferes with glucose transport or utilization. It has been reported in animals and adults that water deprivation and elevated temperatures decrease ketosis.40, 41 Animal studies have shown that acidosis decreases and alkalosis increases ketonemia and ketonuriaW, 4a This is contrary to the impression that one gains from clinical situations in pediatrics, but no studies have been reported. The best study of the effect of fasting ketosis on the serum electrolyte composition is that of Gamble. 44 CHILDREN WITH PREDISPOSITION
A TO
KETOSIS
Weymuller and Schloss4~ studied the response of children who were observed to become markedly ketotic in association with infections. These children developed ketosis more rapidly and profoundly than did normal children when fasted or when given a high-fat diet. When these children were primed for several days with a high-carbohydrate diet and then fasted, they developed marked ketosis more rapidly than the normal child under similar circumstances. When these children were maintained on a high-fat diet for 2 weeks and then fasted for 48 hours, they responded with a decrease of ketosis during the fast and no change in blood sugar. The normal control children showed a heightening of ketosis and a fall of blood sugar under these circumstances. This might be interpreted as evidence for adaptive enzyme formation or adaptive elaboration of regulators in the ketosis-susceptible children. We have studied 2 brothers who developed ketonemia whenever their blood sugar dropped below 100 mg. per cent. This could be effected by a 12 to 14 hour fast. The blood
30 1
sugar and unesterified fatty acid response to epinephrine was normal, whereas response of the ketone bodies was markedly exaggerated. Their growth and development were normal. RESISTANCE TO KETOSIS
OF I N F A N T S
The failure of small infants to develop ketosis when subjected to conditions which would produce a marked ketosis in a child has been a clinical observation of long standing. Even fasting of 9 to 10 days in the infants of less than 9 months does not result in a significant ketosis. 46 Very little attention has been directed toward the elucidation of the cause of this resistance to ketosis in infants. It was shown that the disappearance curve in the serum or concentration in the urine of infants administered acetone, acetoacetate, or fl hydroxybutyrate was similar to that of older children, indicating that the peripheral tissues were able to utilize ketone bodies normallyW, 4s It was further shown that there was no difference in the renal handling of ketone bodies in the infant and the older child. 4s Kaye 49 also demonstrated a normal rise of unesterified fatty acids in infants following the administration of epinephrine. It would appear clear that peripheral utilization and renal handling are vindicated. The fact that unesterified fatty acids are mobilized normally from adipose depots would suggest that mobilization is normal. The epinephrine response tends to indicate, as a first approximation, that the rate of turnover of fatty acids in the serum is also normal. Several possibilities remain to be answered: 1, What role do different pituitary hormones play in this resistance to ketosis? 2, Is there a deficiency of one or more of the agents which stimulate fatty acid oxidation (e.g., carnitine) or some other regulator compound? 3, Could it be that enzymes necessary for the production of ketosis do not appear until the latter part of the first year of life ? The first and third possibilities are readily approachable experimentally.
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February 1962
SUMMARY
T h e biochemical reactions related to the formation of ketone bodies have been reviewed. A n attempt has been made to indicate the metabolic interrelationship of fats, carbohydrates, and hormones. Although there has been notable progress in the field of fat metabolism in the last I0 years, there remain m a n y unanswered questions. I n relation to ketosis the fundamental question is the mechanism of regulation of acetoacetate synthesis. T h e following are some of the factors which need to be considered when studying the mechanisms involved in the accumulation of ketone bodies: 1. T h e mobilization of fatty acids. a. Agents which stimulate fatty acid mobilization and those which inhibit fatty acid release. b. Metabolism of adipose tissue and the relation to the surrounding medium. 2. Fatty acid oxidation. a. Relation of phosphorylation to oxidation by mitochondria in the presence of an increased concentration of fatty acids. b. T h e rate limiting step of oxidation in the Krebs cycle. c. T h e role of reduced nucleotides. d. T h e role of glucose utilization and gluconeogenesis. e. Nature and effect of metabolites which regulate the pathway to acetoacetate specifically. 3. Peripheral utilization. Several clinical conditions associated with ketosis are briefly surveyed, and it is found that the factors noted above vary in degree and in different situations. Attention is directed to the resistance of small infants to ketosis, and the possible mechanisms involved are discussed.
REFERENCES
1. Engel, F. L.: Metabolic Activity of Adipose Tissue, in Kinsell, L. W., editor: Adipose
2. 3. 4.
5. 6. 7.
8.
Tissue as an Organ, Philadelphia, 1961, F. A. Davis Company. Fredrickson, D. S., and Gordon, R. S.: Transport of Fatty Acids, Physiol. Rev. 38: 585, 1958. Dole, V. P.: The Significance of Nonesterified Fatty Acids in Plasma, A. M. A. Arch. Int. Med. 101: 1005, 1958. Fritz, I, B.: Factors Influencing the Rates of Long-Chaln Fatty Acid Oxidation and Synthesis in Mammalian Systems, Physiol. Rev. 41: 52, 1961. Terner, C.: The Formation of Acetoacetate in Homogenates of the Mammary Gland, Biochem. J. 70: 402, 1958. Weinhouse, S., and Millington, R. H.: A Study of Acetoacetate Turnover in Rat Liver Slices in vitro, J. Biol. Chem. 193: 1, 1951. Gould, A., and Coleman, D. C.: Accumulation of Aeetoacetate in Muscle Homogenates From Dystrophic Mice, Biochim. et biophys, acta 47: 422, 1961. Green, D. ]5., and Wakil, S. J.: Enzymatic Mechanisms of Fatty Acid Oxidation and Synthesis, in Block, K., editor: Lipide Metabolism, New York, 1960, John Wiley & Sons, Inc.
9. Campbell, J., and Best, C. H.: Physiologic Aspects of Ketosis, Metabolism 5: 95, 1956. 10. I-Iird, F. J. R., and Symons, R. H.: The Mode of Formation of Ketone Bodies From Butyrate by Tissues From the Rumen and Omasum of the Sheep, Bioehim. et biophys. acta 46: 457, 1961. 11. Bagchi, S. P., Mushahwar, I. K., Chang, T., Koeppe, R. E., and Mourkides, G. A.: Aeetoacetate and Acetate, Intermediates in Glutarate Metabolism, J. Biol. Chem. 236: 370, 1961. 12. Jencks, W. P., Cordes, S., and Larriuolo, J.: The Free Energy of Thiol Ester Hydrolysis, J. Biol. Chem. 235: 3608, 1960. 13. McCann, W. P.: The Oxidation of Ketone Bodies by Mitochondria From Liver and Peripheral Tissues, J. Biol. Chem. 226: 15, 1957. 14. Engel, F. L.: The Influence of the Endocrine Glands on Fatty Acid and Ketone Body Metabolism, A. M. A. Arch. Int. Med. 100: 18, 1957. 15. Jeanrenand, B., and Renold, A. E.: Studies on Rat Adipose Tissue in vitro. VII. Effects of Adrenal Cortical Hormones, J. Biol. Chem. 235: 2217, 1960. 16. Engel, F. L.: Extra-Adrenal Actions of Adrenocorticotropin, Vitamins & Hormones 19:1961 (in press). 17. Vaughan, M.: Effect of Hormones on Phosphorylase Activity in Adipose Tissue, J. Biol. Chem. 235: 3049, 1960. 18. Berthet, J.: Action du glucagon et de l'adr&~aline sur le m~tabolisme des llpldes dans le tissu h~patique. Fourth Int. Congr. Bioehem., Vienna, 1958, p. 107. 19. Langdon, R. G.: Hormonal Regulation of Fatty Acld Metabolism, in Block, K., editor:
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20.
21.
22.
23. 24. 25.
26. 27. 28. 29.
30.
31.
32.
33.
34. 35. 36.
Lipide Metabolism, New York, 1960, John Wiley & Sons, Inc. Lardy, H. A., and Wellman, H.: Oxidative Phosphorylations: Role of Inorganic Phosphate and Acceptor Systems in Control of Metabolic Rates, J. Biol. Chem. 185: 215, 1952. Bierman, E. L., Schwartz, I. L., and Dole, V. P.: Action of Insulin on Release of Fatty Acids From Tissue Stores, Am. J. Physiol. 191: 359, 1957. Raben, M. S., and Hollenberg, C. H.: Effect of Glucose and Insulin in the Esterification of Fatty Acids by Isolated Adipose Tissue, J. Clin. Invest. 39: 435, 1960. Shaw, W. V., and Tapley, D. F.: Oxaloacetate in the Livers of Alloxan-Diabetic Rats, Biochim. et biophys, acta 30: 426, 1958. Siperstein, M. D.: Levels of TCA Cycle Intermediates, J. Clin. Invest. 39: 986, 1960. Lassow, W. J., Brown, G. W., Jr., and Chaikoff, I. L.: The Action of Insulin in Sparing Fatty Acid Oxidation: A Study With Palmitic Aeid-l-C14 and Octonoate-l-C14, J. Biol. Chem. 220: 839, 1956. Lehninger, A. L.: A Quantitative Study of the Product of Fatty Acid Oxidation in Liver Suspensions, J. Biol. Chem. 164: 291, 1946. Krebs, H. A.: The biochemical lesion in Ketosis, A. M. A. Arch. Int. Med. 107: 51, 1961. Menkes, J., and Sidbury, J. B., Jr.: Unpublished data. Gammeltoff, A.: The Significance of Ketone Bodies in the Arterial and Venous Blood in Human Subjects During Starvation, Acta physiol, scandinav. 19: 270, 1950. Chahners, I. M., Pawan, G. L. S., and Kekwick, A.: Fat-Mobilizing and Ketogenic Activity of Urine Extracts: Relation to Corticotrophin and Growth Hormone, Lancet 2: 6, 1950. Glock, G. E., McLean, P., and Whitehead, J. K.: Pathways of Glucose Catabolism in Rat Liver in Alloxan Diabetes and Hyperthyroidism, Biochem. J. 63: 520, 1956. Abraham, S., Matthes, K. J., and Chaikoff, I. L.: Hydrogen Transfer From TritiumLabeled Pyridine Nucleotides to Fatty Acids Synthesized by Homogenate Fractions of Lactating Rat Mammary Gland, Biochim. et biophys, acta 47: 424, 1961. Lowenstein, J. M.: The Pathway of Hydrogen In Biosyntheses. I. Experiments With Glucose1-H3 and Lactate-2-H 3, J. Biol. Chem. 236: 1213, 1961. Engelson, G. E., and Zetterquist, P.: Congenital Diabetes Mellitus and Pseudodiabetes Mellitus, Arch. Dis. Childhood 32: 193, 1957. Kekwiek, A., Pawan, G. L. S., and Chalmers, T. M.: Resistance to Ketosis in Obese Subjects, Lancet 2:1157, 1959. Ross, S. G., and Josephs, H. W.: Observations
Ketosis
37.
38. 39. 40. 41.
42.
43.
44. 45. 46.
47. 48.
49. 50.
51.
52.
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on the Metabolism of Recurrent Vomiting, Am. J. Dis. Child. 28: 447, 1924. Mirsky, I. A., and Nelson, W. E.: Ketosis in Relation to the Hepatic Reserves of Glycogen, A Study of Normal and of Diabetic Children, Am. J. Dis. Child. 67: 100, 1946. Mayes, P. A.: Level of Liver Glycogen in Ketosis, Nature 182: 324, 1958. Salomonsen, L. E., and Harmaes, K.: The Blood Sugar in Cyclic Vomiting, Acta paediat. 47: 383, 1958. Mackay, E. M., Wick, A. N., and Visscher, F. E.: Dehydration and Ketosis, Proc. Soc. Exper. Biol. & Med. 47: 351, 1941. Johnson, R. E., Passmore, R., and Sargent, F., II.: Multiple Factors in Experimental Human Ketosis, A. M. A. Arch. Int. Med. 107: 43, 1961. Mackay, E. M., Wick, A. N., Carne, H. O., and Barnum, C. P.: The Influence of Alkalosis and Acidosis Upon Fasting Ketosis, J. Biol. Chem. 138: 63, 1940. Lipsky, S. R.: Effects of Alkalosis Upon Ketone Body Production and Carbohydrate Metabolism in Man, J. Clin. Invest. 33: 1269, 1954. Gamble, J. L., Ross, G. S., and Tisdall, F. F.: Metabolism of Fixed Bases During Fasting, J. Biol. Chem. 57: 633, 1923. Weymuller, C. A., and Schloss, O. M.: NonDiabetic Ketosis in Children, Am. J. Dis. Child. 34: 549, 1927. Beatty, C. H., Marco, A., Peterson, R. D., Bock, R. M., and West, E. S.: Acetoacetic Acid Metabolism by Skeletal Muscle Fibers From Control and Diabetic Rats, J. Biol. Chem. 235: 2774, 1960. Heymann, W.: Metabolism Studies on Age Disposition to Ketosis in Human Beings, J. PEDIAT. 12: 21, 1938. Williams, M. L , Kaye, R., and Kumagai, M.: Studies on the Mechanism of Ketosis in Infants and Children, A. M. A. J. Dis. Child. 94: 499, 1957. Kaye, R., and Kumagai, M.: Studies of Unesterified Fatty Acid Metabolism in Infants, A. M. A. J. Dis. Child. 96: 527, 1958. Chalmers, I. M., Pawan, G. L. S., and Kekwick, A.: Fat-Mobilizing and Ketogenic Activity of Urine Extracts: Relation to Corticotrophin and Growth Hormone, Lancet 2: 6, 1960. Rudman, D., DiGiolamo, M., Kendall, F. E., Werthein, A. R., Seidman, F., Reid, M. B., and Bern, S.: Further Observations on the Effect of Pituitary Gland Extracts Upon the Serum of Lipids of the Rabbit, Endocrinology 67: 784, 1960. Seifter, J., and Baeder, D. H.: Lipid Mobilizer (LM) From Posterior Pituitary of Hogs, Proc. Soc. Exper. Biol. & Med. 94: 318, 1957.
Ketosis in infants and children
j. B. Sidbury, Jr., M.D., * and Boek L. Dong, M.D. BALTIMORE,, MD.
albumin,2, a are then transported to the tissues of the body for metabolism. Transported fatty acids are utilized by many of the cells of the body as a metabolic fuel; the brain is a notable exception. * Acetoacetate is one of the products of fatty acid oxidation. Acetone and fl hydroxybutyrate are formed from acetoacetate. 4 Although acetoacetate formation has been demonstrated in the lactating mammary gland, 5 kidney, ~ and muscle7 blood ketone bodies are derived from the liver. It should be noted that ketone bodies are present in the blood of normal persons and that ketosis represents an accumulation of normal metabCites. The fatty acids which are transported from adipose tissue via serum albumin are taken up by the liver and may either be synthesized into triglycerides or may be sequentially oxidized, 2 carbon fragments at a time as originally suggested by Knoop. s This is effected by activation of the fatty acid with acetyl eoenzyme A. The oxidation of fatty acids occurs exclusively in the mitochondria of the cell. The enzymatic path-
K E T O S I S is a condition which is frequently encountered by those treating children. It is a manifestation of altered metabolism and is almost invariably associated with some other disease process. Ketosis is frequently associated with diabetes, fasting, diarrhea, vomiting, and glycogen storage disease. Some patients are quite prone to develop ketosis and do so with any febrile illness. The particular interest of the pediatrician is based on the greater susceptibility of children and on the intriguing paradox that young infants are more resistant to ketosis than either the child or the adult. The first step in the elaboration of ketone bodies occurs in the adipose tissue, where a lipase hydrolyzes the individual fatty acids from the depot form of fat, triglycerides?, 2 The nonesterified fatty acids, bound to serum From the Department of Pediatrics, The Johns Hopkins University School o[ Medicine and the Johns Hopkins Hospital. This work was supported in part by Grant B-1626 from The National Institutes of Health, Department of Health, Education and Welfare. *Presen~ Address, Department o[ Pediatrics, Duke University School o[ Medicine, Durham, N. C.
294
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Ketosis
ways for oxidation and synthesis are distinct.k, s We have indicated that the ketone bodies found in the blood arise principally from the liver. Acetoacetate is the ketone body first produced by the liver, and the other 2 are derived from it 9 (Fig. 1). The explanation of the fact that the liver is the site of origin of the ketone bodies of blood lies in the difference in the pattern of enzymes in the liver and peripheral tissues (vide infra). The following reactions are pertinent. ~ Acetoacetyl-CoA synthesizing reactions: Thiolase >
1. 2AcCoA < AcAcCoA + CoA Aeetoacetate activating enzyme > 2. AcAc + CoA + ATP < AcAcCoA + AMP + PP (See also reaction 5.) Formation of acetoacetate: Deacylase 3. AcAcCoA + H20 > AcAc + CoA HMG-CoA condensing enzyme >
4. AcAcCoA + AcCoA < 3 hydroxy3-methylglutaryl-CoA Cleavage enzyme HMG-CoA > AcAc + AcCoA CoA transferase >
5. AcAcCoA + Succinate < Succinyl CoA + AcAe Aeetoacetyl glutathione thioesterase >
6. AcAcCoA + GSH < CoA AcAc -SG + H20
AcAc -SG + > AcAc + GSH
~Abbreviatlons used: AcAc, acetoacetate; AcCoA, acetyl coenzyme A; AcAcCoA, acetoacetyl eoenzyme A; /3Oit, flhydroxybutyrate; AMP, adenosine monophosphate; ADP, adenosine dlphosphate; ATP, adenosine triphosphate; DPN, DPNH, dlphosphopyrldine nueleotide, oxidized and reduced; TPN, TPNH, triphosphopyridine nudeotlde, oxidized and reduced; GSH, reduced glutathlone; HMP, hexose monophosphate shunt; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CoA, coenzyme A; UFA, unesterified fatty adds; ACTH, adrenocorticotrophle hormone; TSH, thyroid stimulating hormone.
295
There is no agreement on whether acetoacetate is formed mainly by reaction 3 or 4. There is no conclusive evidence to support one theory rather than the other. Recently, a study of ketone body formation in the tissues from the rumen and omasum of the sheep with the use of C ~4 butyrate suggests that the acetoacetate formed was derived by HMP-CoA. 1~ For the present, a final judgment should be withheld until it can be determined to what extent in vitro conditions determine the resultant labeling and hence the conclusions. Leucine has been shown to be converted to acetate by way of acetoacetate? ~ Reactions 2 and 5 do not occur in the liver. The other reactions are present in the liver and in the peripheral tissues. Reaction 6 is postulated--not p r o v e d - - b u t it could occur nonenzymatically. TM Since reactions 2 and 5 are absent in the liver, acetoacetate, once formed, must pass into the circulating blood to be metabolized by the peripheral tissues. A certain portion is converted into fiOH before it leaves the liver: fl hydroxybutyrate dehydrogenase 7. AcAc + DPNH < flOH + DPN Thus the liver possesses the enzymes necessary for forming acetoacetate and flOH but lacks an enzyme necessary for their utilization. The peripheral tissues have the enzymes necessary for their utilization. The second phase of ketone metabolism to consider is utilization. It is apparent from the above discussion that, once formed in the liver, ketone bodies are utilized b y other tissues. It is assumed that flOH is used by the tissues through the reverse reaction of flOH dehydrogenase and then esterification of the acetoacetate with coenzyme A via the thiolase or the coenzyme A transferase reactions. 13 HORMONAL KETOSIS
EFFECTS
ON
A particular hormone may demonstrate an effect in adipose tissue which is conducive to ketosis but the effect induced in the liver
2 9 6 Sidbury and Dong
is antiketogenic (e.g., cortisol); therefore, the effect of a hormone in the intact animal and in isolated tissues in vitro may be opposite (Table I). Virtually all of the hormones affect adipose tissue either directly or indirectly? It will be immediately apparent that a given hormone may either have no effect on adipose tissue, may stimulate glucose uptake, and inhibit fatty acid release, or it may promote the release of fatty acids. Cortisone and cortisol suppress the ketosis associated with cold stress and fluoroacetate (a Krebs cycle poison), but they have no effect on ketosis due to insulin hypoglycemia or administration of pituitary extract? 4 The antiketogenic effect of glucocorticoids is due in part to the fact that they stimulate fat synthesis and gluc0neogenesis. Cortisol effects a release of fatty acids from adipose tissue in vitro2 5 Epinephrine, norepinephrine, ACTH, growth hormone, and glucagon cause in vitro and in vivo fatty acid mobilization under certain conditions and an increase in ketone body production in vivo. 1, 18 Epinephrine has been shown to activate phosphorylase by stimulating the synthesis of 3', 5' adenylic acid9 7 and recently the latter has been shown to have a direct ketogenic elfeet? s The association of insulin deficiency and ketosis is the hormonal relation that stands out in the minds of everyone. Not only has ketosis long been established as a feature of diabetes, but it is recognized as a sign of poor control--a warning sign. A great deal of study has been undertaken to determine the relation of insulin to excess ketonemia. A deficiency of insulin in the diabetic animal and man has been shown to be associated with increased serum unesterified fatty acid levels, increased acetoacetate formation from fatty acids, and markedly diminished fatty acid synthesis. Simultaneously, the deprivation of insulin results in an elevation of blood glucose, an increased rate of hepatic gluconeogenesis, decreased glucose utilization, decreased rate of incorporation of amino acids into protein, and increased total body oxygen consumption. The administration of in-
February 1962
snlin is associated with a decrease in serum unesterified fatty acids, decrease in acetoacetate formation, and resumption of normal fatty acid synthesis. It suppresses acetoacetate synthesis from fatty acids more than the oxidation to carbon dioxide. The effect of insulin is not apparent on short-chain fatty acids? 9 On t h e other hand insulin in excess induces ketosis. The mechanism envisaged in this situation is that homeostatic mechanisms are brought to bear which, broadly speaking, are directed toward returning the blood sugar to normal; and as a corollary glucose utilization is reduced to a minimum, and other substrates are used either as an energy source or as a carbohydrate source. Thus, proteins (amino acids) are converted to glucose, and fatty acids are mobilized at an accelerated rate. It is postulated that epinephrine is released during hypoglycemia and may he important in these reactions? 4 Thus, the hormones which favor the development of ketosis uniformly stimulate fatty acid mobilization from the fat depots, suppress the respiratory quotient, reduce glucose utilization, and frequently inhibit insulin action. METABOLIC IN KETOSIS
INTERRELATIONS
A very important consideration, necessary to the understanding and evaluation of ketosis in any particular situation, is the relationship of fatty acid oxidation and synthesis to the Krebs cycle and to glucose metabolism. We have attempted to illustrate these interrelations in Fig. 2. It has been emphasized that fatty acid oxidation results in the formation of one acetyl coenzyme A molecule for each pair of carbon atoms of the fatty acid. As indicated in Fig. 2, acetyl coenzyme A has a number of routes open to it. It may condense with oxaloacetate to form citrate and be oxidized to COs and water through the cycle; this is probably the major pathway. The oxidation of acetate via the Krebs cycle is an energy generating sequence of events. On the other hand, acetyl coenzyme
Volume 60, Number 2
Ketoszs
Table I. Effect of hormones on adipose tissue r Promote glucose uptake and inhibit fatty acid release Insulin Prolacfin
Stimulate fatty acid release Epinephrine Norepinephrine ACTH TSH Growth hormone Glucagon Lipid mobilizing factors (Chalmers, 5o Rudman, 5v Seifter 52) Cortisol
~'After E,ngel.1
A may form acetoacetyl coenzyme A which can proceed to cholesterol synthesis, fatty acid synthesis, or ketone body formation. The synthesis of both fatty acids and cholesterol requires the reduced nucleotide, T P N H . T h e factors regulating the direction in which acetoacetate coenzyme A proceeds are not clear. Acceleration of acetoacetate to fl hydroxybutyrate by certain substrates has been explained by increased supply of reduced DPN. 4 iIt has been shown that, under the circumstances, the rate of oxidation in the mitochondria may be limited by the process of phosphorylation. 2~ The latter requires an adequate and available supply of ADP. Utilization of glucose involves phosphorylation by ATP, and ADP is a product. Thus the regeneration of ADP for oxidative phosphorylation may be one way in which glucose "spares" fat oxidation. Another potential role of glucose in fat metabolism is the generation of T P N H by the oxidation of glucose6-phosphate through the hexose monophosphate shunt. TPNI-t has been shown to be necessary for cholesterol and fatty acid synthesis. Glucose also spares fatty acid oxidation by decreasing the release of fatty acids from adipose tissues 21 and increasing incorporation of fatty acids into glycerides.22 OVERPRODUCTION VERSUS UNDERUTILIZATION
It would seem apparent that increased levels of ketone bodies in the blood and
29 7
urine would of necessity have to result from either an increased production of these products or their parent metabolites or a decreased rate of their catabolism. All evidence points to a markedly increased rate of mobilization and production of fatty acids in those conditions associated with ketosis. Thus overproduction of the metabolites which are the source of ketone bodies is present. The question remaining is whether under-utilization in any way contributes to ketosis. This proposition can be divided into three phases of inquiry. Whether or not there is a decreased level of oxaloacetate and hence the rate of oxidation of fatty acids is secondarily decreased is an old hypothesis. The evidence at hand indicates that there is no decrease in oxaloacetate either in diabetic or fasting ketosis. TM 24 Furthermore, it is clear that there is in fact an increased rate of oxidation of fatty acids in those conditions associated with ketosis. 25 The next question asked was if Krebs cycle intermediates are added, would the rate of oxidation of fatty acids via the Krebs cycle increase and the formation of acetoacetate decrease? With the proper in vitro conditions Krebs cycle intermediates can be shown to decrease acetoacetate formation. 2~ In addition Krebs cycle inhibitors both in vivo and in vitro .result in the accumulation of acetoacetate?4, 2,. 28 The response to the administration of Krebs cycle intermediates to an animal with ketosis has given rise to conflicting reports. A fundamental consideration here is whether under the conditions conducive to ketosis the Krebs cycle is operating at a maximal rate. In any series of reactions one encounters a slow step, relative to the rates of the other reactions. It is not possible to answer this question at the present time. It is clear that K the rate of oxidation of fatty acids by way of the Krebs cycle could be increased there would be a lessened production of acetoacetate. The second consideration in underutilization is the rate of peripheral utilization. One must assume that the rate of utilization of ketone bodies by the peripheral tissues is proceeding at a near maximal rate for the
2 98
Sidbury and Dong
OPNH AcCoA ~
AcAcCoA
.
DPN
February 1962
BOHCoA ,r
~.
CaOTONYLCoA
"~ F.A.
DPId89
LEUCINE ~
BOH
H MG CoA ~
AC AC ~
CHOLESTEROL
TYROSINE ACETONE
1 pHENYLALANINE
Fig, 1, This schema illustrates the pathways of origin of acetoacetate from acetyl CoA (AcGoA), leucine, and phenylalanine. T h e derivation of the other 2 ketone bodies, acetone and fl hydroxybutyrate ( # O H ) , from acetoacetate is indicated. The reaction in fatty acid synthesis and cholesterol synthesis in mitochondria requiring the reduced nucleotide T P N H is noted.
G;ycogen
I GlucoseI - P ~ ATP ADP
II
UDPG
UTP
PP
Fructose6-P
~OH
T~ Fruciose 1,6-P 11
t~
Tr,oso,
HMG-CoA -
2 DPNH + 2ATP-~-"ii coz + ADP P- Pyruvote~
/ II ~e",/
II
- I " ~ . . J ' ' X
Amino ocid~DPNH~r~[; Pyruvote + CO;:TPNH~TPN M ~ e
OPN"~"]}" Lgclote
Eotfy acids
" Ac-Ac-CoA |
TP,.,,. ~I It
) Acetylotion .... ~Amino acids
Y/)~I-TPN'~,-TPNH
Su~cc ~L Ketoglufarofe inYI- CoA~/~LCOz
Fig. 2. This metabolic map illustrates some of the known and possible interrelationships relating to acetyl-CoA metabolism. T h e reactions involving nucleotide coenzymes are indicated. T h e reactions of the Krebs cycle and those immediate to AcCoA are catalyzed in or on the mitochondria; the other reactions (glycolysis and the H M P shunt) occur in the nonparticulate portion of the cell.
Volume 60 Number 2
substrate level remains high. It is possible then that the accumulation in the blood is due to a reduced rate of utilization by the peripheral tissues. The evidence is to the contrary. The peripheral tissues are utilizing acetoacetate and flOH at a greatly increased rate during ketosis. 29 The third consideration in underutilization is regarded under this heading with misgivings. The question of metabolic regulators, or naively state metabolic compounds which direct traffic, is pertinent but has received little attention. Here we have in mind substances which would act to increase the formation of acetoacetate, though the regulator is not a direct participant or substrate in the reaction. Too little attention has been given this consideration to adduce a compelling case for it. ALTERATION LEVELS
IN ENZYME
It has recently been demonstrated that the activity of one or more of the enzyme systems concerned with formation of aeetoacetate from acetoacetate coenzyme A is increased in the liver of the diabetic rat2 ~ It is proposed that this may be the mechanism for ketosis in diabetes. However, since it has been shown that infusion of oetonoate results in a marked increase in ketone body formation in the normal individual, 14 this would suggest that the enzyme systems concerned with the reaction of acetoacetyl coenzyme A to acetoacetate are not the rate limiting steps. It appears more likely that the observed increase in enzyme activity is an effect rather than a cause, similar to the increase in glucose-6-phosphatase activity which has also been observed in the diabetic rat. 19 Glock and McLean 31 have found a decreased level of glucose-6-phosphate dehydrogenase in the liver of diabetic rats. The importance of this finding relates to the question of the role of T P N H generated by the hexose monophosphate shunt, as opposed to that generated by the isocitric dehydrogenase reaction, in the regulation of fatty acid synthesis. Recent work supports the
Ketosis
299
primary role of the H M P shunt in fatty acid synthesisY 2 On the other hand, another laboratory has reported the surprising finding that the hydrogen in position 2 of lactate was more efficiently incorporated into synthesized fatty acids in lactating mammary gland slices than was hydrogen from position 1 of glucose. The latter would be donated to T P N H by way of the H M P shunt. A mechanism is postulated. 33 PRECIS
Ketosis, then, results when fatty acids are mobilized from the adipose depots and presented to the liver for metabolism at a rate greater than the capacity of the liver to oxidize them. The overflow, as it were, goes into formation of acetoaeetate which in turn gives rise to acetone and fl hydroxybutyrate. The condition which generally prevails when ketosis arises is that of tissue carbohydrate deprivation. In fasting there is a deprivation of the intake of carbohydrate; in diabetes the carbohydrate bathing the cells cannot gain entrance to the cell. A number of hormones stimulate fatty acid release from adipose depots. Insulin, which inhibits fatty acid mobilization, is decreased in diabetes and probably in fasting. The precise manner in which glucose affects the production of aeetoacetate, and the nature and role of various regulators of acetoacetate formation are not clearly delineated. DIABETES
For our purposes diabetes can be simply described as a condition in which there are metabolic aberrations secondary to a deficiency of insulin relative to the cellular requirements for normal metabolism. The cells are deprived of glucose due to an interference of glucose transport. Since glu. cose metabolism is severely depressed, the cells must of necessity utilize fatty acids as the source of energy under these circumstances. It has indeed been found that the level of serum fatty acids is markedly elevated in the diabetes of the untreated indi-
3 O0 Sidbury and Dong
February 1962
vidual or the diabetic in ketosisY Fatty acids are mobilized at a rate in excess of the ability of the liver to oxidize them and the excess is diverted into ketone body formation. This is not totally wasteful for the peripheral tissues utilize the major proportion of the ketone bodies formed as an energy source, and only a small fraction is excreted into the urine. The amount in the urine is significant quantitatively; therefore, the over-all process is quite inefficient. It is of interest that ketosis is not as universally associated with diabetes in the infant as it is in the juvenile patient with diabetesY ~ Ketosis in the patient with diabetes.indicates the degree of insulin deprivation and the extent to which the metabolism has been altered. FASTING
KETOSIS
It would seem reasonable, in the light of the experimental evidence available, to conelude that basically the mechanisms operative in the production of ketosis are similar in ketosis due to fasting. The difference would be one of degree and the particular. This unified view results from ignorance in part, and the similarities may be found to be more apparent than real when the factors involved in regulation are appreciated. There seems little doubt that there is an increased mobilization of fatty acids which exceeds the capability of oxidation by the liver. OBESITY
It has been observed that obese adults are markedly less susceptible to fasting ketosis than are normal or thin adults2 ~ The very occasional observation in obese children suggests that this finding is peculiar to adults. GLYCOGEN
STORAGE
DISEASE
Certain forms of glycogen storage disease are also associated with ketosis. The types which are associated with ketosis are those associated with an absence of glucose-6phosphatase, decreased amylo-l,6-glucosidase (limit dextrinosis), and those associated with decreased phosphorylase activity in the
liver. It is of interest that those persons who tend to be more severely ketotic have a marked decrease in ketonemia in a postabsorptive state, but the level is rarely found to be normal in spite of a normal blood sugar. It is of particular interest that patients with glucose-6-phosphatase deficiency represent one of the few conditions that manifest ketosis in the neonatal period. In the first and third types of glycogenosis mentioned there is no reason to believe that muscle metabolism should be altered; hence, it seems unlikely that decreased utilization of ketone bodies by muscle plays a significant role in ketonemia exhibited by these patients. It seems pertinent that in patients with a glucose-6-phosphatase defect there is a large excess of mobilizable glycogen, but ketosis is a prominent feature of the clinical syndrome. The liver could hardly be considered deficient of glucose-6-phosphate. This observation increases the enigma of the mechanism of the ketolytic effect of glucose, suggesting perhaps that the suppressive effect of glucose on fatty acid release from adipose depots is the more important of its postulated roles. KETOSIS ASSOCIATED WITH VOMITING AND DIARRHEA
In the practice of pediatrics, one encounters ketosis most frequently in association with vomiting and diarrhea. The mechanisms involved have not been studied. The association is seen most dramatically in the condition described as recurrent or cyclic vomiting. Some of these patients have a marked hypoglycemia; therefore, the mechanism invoked for fasting ketosis would be applicable? 6 In addition, the general nutrition of these patients is usually very poor, and it would be reasonable to suppose that their liver glycogen supply is below normal and the protein available for gluconeogenesis is minimal; this explains the rapidity with which they go into ketosis. A relation between the degree of ketosis and a reduction in liver glycogen has been shown, but it is not primary to the induction of ketosis. ~7, 3~ Some patients with cyclic vomiting have a
Volume 60 Number 2
Ketosis
normal blood sugar level, and others have been described with a high level? 9 The latter might be considered to be associated with a transient or nutritionally induced hypoinsulinism. The primary metabolic alteration which effects decreased glucose utilization in patients with vomiting and diarrhea is not clear. One could postulate that cellular dehydration interferes with glucose transport or utilization. It has been reported in animals and adults that water deprivation and elevated temperatures decrease ketosis.40, 41 Animal studies have shown that acidosis decreases and alkalosis increases ketonemia and ketonuriaW, 4a This is contrary to the impression that one gains from clinical situations in pediatrics, but no studies have been reported. The best study of the effect of fasting ketosis on the serum electrolyte composition is that of Gamble. 44 CHILDREN WITH PREDISPOSITION
A TO
KETOSIS
Weymuller and Schloss4~ studied the response of children who were observed to become markedly ketotic in association with infections. These children developed ketosis more rapidly and profoundly than did normal children when fasted or when given a high-fat diet. When these children were primed for several days with a high-carbohydrate diet and then fasted, they developed marked ketosis more rapidly than the normal child under similar circumstances. When these children were maintained on a high-fat diet for 2 weeks and then fasted for 48 hours, they responded with a decrease of ketosis during the fast and no change in blood sugar. The normal control children showed a heightening of ketosis and a fall of blood sugar under these circumstances. This might be interpreted as evidence for adaptive enzyme formation or adaptive elaboration of regulators in the ketosis-susceptible children. We have studied 2 brothers who developed ketonemia whenever their blood sugar dropped below 100 mg. per cent. This could be effected by a 12 to 14 hour fast. The blood
30 1
sugar and unesterified fatty acid response to epinephrine was normal, whereas response of the ketone bodies was markedly exaggerated. Their growth and development were normal. RESISTANCE TO KETOSIS
OF I N F A N T S
The failure of small infants to develop ketosis when subjected to conditions which would produce a marked ketosis in a child has been a clinical observation of long standing. Even fasting of 9 to 10 days in the infants of less than 9 months does not result in a significant ketosis. 46 Very little attention has been directed toward the elucidation of the cause of this resistance to ketosis in infants. It was shown that the disappearance curve in the serum or concentration in the urine of infants administered acetone, acetoacetate, or fl hydroxybutyrate was similar to that of older children, indicating that the peripheral tissues were able to utilize ketone bodies normallyW, 4s It was further shown that there was no difference in the renal handling of ketone bodies in the infant and the older child. 4s Kaye 49 also demonstrated a normal rise of unesterified fatty acids in infants following the administration of epinephrine. It would appear clear that peripheral utilization and renal handling are vindicated. The fact that unesterified fatty acids are mobilized normally from adipose depots would suggest that mobilization is normal. The epinephrine response tends to indicate, as a first approximation, that the rate of turnover of fatty acids in the serum is also normal. Several possibilities remain to be answered: 1, What role do different pituitary hormones play in this resistance to ketosis? 2, Is there a deficiency of one or more of the agents which stimulate fatty acid oxidation (e.g., carnitine) or some other regulator compound? 3, Could it be that enzymes necessary for the production of ketosis do not appear until the latter part of the first year of life ? The first and third possibilities are readily approachable experimentally.
3 0 2 Sidbury and Dong
February 1962
SUMMARY
T h e biochemical reactions related to the formation of ketone bodies have been reviewed. A n attempt has been made to indicate the metabolic interrelationship of fats, carbohydrates, and hormones. Although there has been notable progress in the field of fat metabolism in the last I0 years, there remain m a n y unanswered questions. I n relation to ketosis the fundamental question is the mechanism of regulation of acetoacetate synthesis. T h e following are some of the factors which need to be considered when studying the mechanisms involved in the accumulation of ketone bodies: 1. T h e mobilization of fatty acids. a. Agents which stimulate fatty acid mobilization and those which inhibit fatty acid release. b. Metabolism of adipose tissue and the relation to the surrounding medium. 2. Fatty acid oxidation. a. Relation of phosphorylation to oxidation by mitochondria in the presence of an increased concentration of fatty acids. b. T h e rate limiting step of oxidation in the Krebs cycle. c. T h e role of reduced nucleotides. d. T h e role of glucose utilization and gluconeogenesis. e. Nature and effect of metabolites which regulate the pathway to acetoacetate specifically. 3. Peripheral utilization. Several clinical conditions associated with ketosis are briefly surveyed, and it is found that the factors noted above vary in degree and in different situations. Attention is directed to the resistance of small infants to ketosis, and the possible mechanisms involved are discussed.
REFERENCES
1. Engel, F. L.: Metabolic Activity of Adipose Tissue, in Kinsell, L. W., editor: Adipose
2. 3. 4.
5. 6. 7.
8.
Tissue as an Organ, Philadelphia, 1961, F. A. Davis Company. Fredrickson, D. S., and Gordon, R. S.: Transport of Fatty Acids, Physiol. Rev. 38: 585, 1958. Dole, V. P.: The Significance of Nonesterified Fatty Acids in Plasma, A. M. A. Arch. Int. Med. 101: 1005, 1958. Fritz, I, B.: Factors Influencing the Rates of Long-Chaln Fatty Acid Oxidation and Synthesis in Mammalian Systems, Physiol. Rev. 41: 52, 1961. Terner, C.: The Formation of Acetoacetate in Homogenates of the Mammary Gland, Biochem. J. 70: 402, 1958. Weinhouse, S., and Millington, R. H.: A Study of Acetoacetate Turnover in Rat Liver Slices in vitro, J. Biol. Chem. 193: 1, 1951. Gould, A., and Coleman, D. C.: Accumulation of Aeetoacetate in Muscle Homogenates From Dystrophic Mice, Biochim. et biophys, acta 47: 422, 1961. Green, D. ]5., and Wakil, S. J.: Enzymatic Mechanisms of Fatty Acid Oxidation and Synthesis, in Block, K., editor: Lipide Metabolism, New York, 1960, John Wiley & Sons, Inc.
9. Campbell, J., and Best, C. H.: Physiologic Aspects of Ketosis, Metabolism 5: 95, 1956. 10. I-Iird, F. J. R., and Symons, R. H.: The Mode of Formation of Ketone Bodies From Butyrate by Tissues From the Rumen and Omasum of the Sheep, Bioehim. et biophys. acta 46: 457, 1961. 11. Bagchi, S. P., Mushahwar, I. K., Chang, T., Koeppe, R. E., and Mourkides, G. A.: Aeetoacetate and Acetate, Intermediates in Glutarate Metabolism, J. Biol. Chem. 236: 370, 1961. 12. Jencks, W. P., Cordes, S., and Larriuolo, J.: The Free Energy of Thiol Ester Hydrolysis, J. Biol. Chem. 235: 3608, 1960. 13. McCann, W. P.: The Oxidation of Ketone Bodies by Mitochondria From Liver and Peripheral Tissues, J. Biol. Chem. 226: 15, 1957. 14. Engel, F. L.: The Influence of the Endocrine Glands on Fatty Acid and Ketone Body Metabolism, A. M. A. Arch. Int. Med. 100: 18, 1957. 15. Jeanrenand, B., and Renold, A. E.: Studies on Rat Adipose Tissue in vitro. VII. Effects of Adrenal Cortical Hormones, J. Biol. Chem. 235: 2217, 1960. 16. Engel, F. L.: Extra-Adrenal Actions of Adrenocorticotropin, Vitamins & Hormones 19:1961 (in press). 17. Vaughan, M.: Effect of Hormones on Phosphorylase Activity in Adipose Tissue, J. Biol. Chem. 235: 3049, 1960. 18. Berthet, J.: Action du glucagon et de l'adr&~aline sur le m~tabolisme des llpldes dans le tissu h~patique. Fourth Int. Congr. Bioehem., Vienna, 1958, p. 107. 19. Langdon, R. G.: Hormonal Regulation of Fatty Acld Metabolism, in Block, K., editor:
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20.
21.
22.
23. 24. 25.
26. 27. 28. 29.
30.
31.
32.
33.
34. 35. 36.
Lipide Metabolism, New York, 1960, John Wiley & Sons, Inc. Lardy, H. A., and Wellman, H.: Oxidative Phosphorylations: Role of Inorganic Phosphate and Acceptor Systems in Control of Metabolic Rates, J. Biol. Chem. 185: 215, 1952. Bierman, E. L., Schwartz, I. L., and Dole, V. P.: Action of Insulin on Release of Fatty Acids From Tissue Stores, Am. J. Physiol. 191: 359, 1957. Raben, M. S., and Hollenberg, C. H.: Effect of Glucose and Insulin in the Esterification of Fatty Acids by Isolated Adipose Tissue, J. Clin. Invest. 39: 435, 1960. Shaw, W. V., and Tapley, D. F.: Oxaloacetate in the Livers of Alloxan-Diabetic Rats, Biochim. et biophys, acta 30: 426, 1958. Siperstein, M. D.: Levels of TCA Cycle Intermediates, J. Clin. Invest. 39: 986, 1960. Lassow, W. J., Brown, G. W., Jr., and Chaikoff, I. L.: The Action of Insulin in Sparing Fatty Acid Oxidation: A Study With Palmitic Aeid-l-C14 and Octonoate-l-C14, J. Biol. Chem. 220: 839, 1956. Lehninger, A. L.: A Quantitative Study of the Product of Fatty Acid Oxidation in Liver Suspensions, J. Biol. Chem. 164: 291, 1946. Krebs, H. A.: The biochemical lesion in Ketosis, A. M. A. Arch. Int. Med. 107: 51, 1961. Menkes, J., and Sidbury, J. B., Jr.: Unpublished data. Gammeltoff, A.: The Significance of Ketone Bodies in the Arterial and Venous Blood in Human Subjects During Starvation, Acta physiol, scandinav. 19: 270, 1950. Chahners, I. M., Pawan, G. L. S., and Kekwick, A.: Fat-Mobilizing and Ketogenic Activity of Urine Extracts: Relation to Corticotrophin and Growth Hormone, Lancet 2: 6, 1950. Glock, G. E., McLean, P., and Whitehead, J. K.: Pathways of Glucose Catabolism in Rat Liver in Alloxan Diabetes and Hyperthyroidism, Biochem. J. 63: 520, 1956. Abraham, S., Matthes, K. J., and Chaikoff, I. L.: Hydrogen Transfer From TritiumLabeled Pyridine Nucleotides to Fatty Acids Synthesized by Homogenate Fractions of Lactating Rat Mammary Gland, Biochim. et biophys, acta 47: 424, 1961. Lowenstein, J. M.: The Pathway of Hydrogen In Biosyntheses. I. Experiments With Glucose1-H3 and Lactate-2-H 3, J. Biol. Chem. 236: 1213, 1961. Engelson, G. E., and Zetterquist, P.: Congenital Diabetes Mellitus and Pseudodiabetes Mellitus, Arch. Dis. Childhood 32: 193, 1957. Kekwiek, A., Pawan, G. L. S., and Chalmers, T. M.: Resistance to Ketosis in Obese Subjects, Lancet 2:1157, 1959. Ross, S. G., and Josephs, H. W.: Observations
Ketosis
37.
38. 39. 40. 41.
42.
43.
44. 45. 46.
47. 48.
49. 50.
51.
52.
303
on the Metabolism of Recurrent Vomiting, Am. J. Dis. Child. 28: 447, 1924. Mirsky, I. A., and Nelson, W. E.: Ketosis in Relation to the Hepatic Reserves of Glycogen, A Study of Normal and of Diabetic Children, Am. J. Dis. Child. 67: 100, 1946. Mayes, P. A.: Level of Liver Glycogen in Ketosis, Nature 182: 324, 1958. Salomonsen, L. E., and Harmaes, K.: The Blood Sugar in Cyclic Vomiting, Acta paediat. 47: 383, 1958. Mackay, E. M., Wick, A. N., and Visscher, F. E.: Dehydration and Ketosis, Proc. Soc. Exper. Biol. & Med. 47: 351, 1941. Johnson, R. E., Passmore, R., and Sargent, F., II.: Multiple Factors in Experimental Human Ketosis, A. M. A. Arch. Int. Med. 107: 43, 1961. Mackay, E. M., Wick, A. N., Carne, H. O., and Barnum, C. P.: The Influence of Alkalosis and Acidosis Upon Fasting Ketosis, J. Biol. Chem. 138: 63, 1940. Lipsky, S. R.: Effects of Alkalosis Upon Ketone Body Production and Carbohydrate Metabolism in Man, J. Clin. Invest. 33: 1269, 1954. Gamble, J. L., Ross, G. S., and Tisdall, F. F.: Metabolism of Fixed Bases During Fasting, J. Biol. Chem. 57: 633, 1923. Weymuller, C. A., and Schloss, O. M.: NonDiabetic Ketosis in Children, Am. J. Dis. Child. 34: 549, 1927. Beatty, C. H., Marco, A., Peterson, R. D., Bock, R. M., and West, E. S.: Acetoacetic Acid Metabolism by Skeletal Muscle Fibers From Control and Diabetic Rats, J. Biol. Chem. 235: 2774, 1960. Heymann, W.: Metabolism Studies on Age Disposition to Ketosis in Human Beings, J. PEDIAT. 12: 21, 1938. Williams, M. L , Kaye, R., and Kumagai, M.: Studies on the Mechanism of Ketosis in Infants and Children, A. M. A. J. Dis. Child. 94: 499, 1957. Kaye, R., and Kumagai, M.: Studies of Unesterified Fatty Acid Metabolism in Infants, A. M. A. J. Dis. Child. 96: 527, 1958. Chalmers, I. M., Pawan, G. L. S., and Kekwick, A.: Fat-Mobilizing and Ketogenic Activity of Urine Extracts: Relation to Corticotrophin and Growth Hormone, Lancet 2: 6, 1960. Rudman, D., DiGiolamo, M., Kendall, F. E., Werthein, A. R., Seidman, F., Reid, M. B., and Bern, S.: Further Observations on the Effect of Pituitary Gland Extracts Upon the Serum of Lipids of the Rabbit, Endocrinology 67: 784, 1960. Seifter, J., and Baeder, D. H.: Lipid Mobilizer (LM) From Posterior Pituitary of Hogs, Proc. Soc. Exper. Biol. & Med. 94: 318, 1957.