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Measurement of urinary lactic, 3-hydroxybutyric, pyruvic and acetoacetic acids in a single analysis using selected ion monitoring and stable isotope labelling techniques.

Sodium borodeuteride treatment of urine expected to have large concentrations of pyruvic and acetoacetic acids produces lactic and 3-hydroxybutyric acids labelled with deuterium. Mass fragmentographic techniques applied to the trimethylsilyated extract of the urine spiked with more extensively labelled analogues as internal standards permit the reliable determination of lactic, pyruvic, 3-hydroxybutyric and acetoacetic acids in a single analysis.

3-Hydroxybutyric Acid↗

Adjuvant effects of glyceryl esters of acetoacetic acid on rectal absorption of insulin and inulin in rabbits.

The promoting effect of glyceryl esters of acetoacetic acid on the rectal absorption of insulin and inulin was studied. A decrease in the serum glucose level was observed in rabbits following the administration of an insulin suppository containing glyceryl-1,3-diacetoacetate (adjuvant II) or 1,2-isopropylideneglycerine-3-acetoacetate (adjuvant IV). The promoting effects of adjuvants II and IV on the rectal absorption of insulin and inulin were suppressed by the addition of calcium and magnesium to the suppository. This indicates that adjuvant interaction with the calcium and magnesium ion located in the rectal membrane is involved in the enhanced absorption of insulin and inulin. Adjuvant release from the suppository formulation in addition to adjuvant lipid solubility were found to be other important factors for enhanced absorption of insulin and inulin.

Acetoacetates↗

Succinyl-CoA:acetoacetate transferase deficiency: identification of a new patient with a neonatal onset and review of the literature.

UNLABELLED: We describe the clinical symptoms and biochemical findings of a patient with succinyl-CoA:acetoacetate transferase deficiency who presented in the neonatal period and review the current literature on this subject. Our patient was initially suspected to have distal renal tubular acidosis, and subsequently, a fasting test revealed severe metabolic ketoacidosis with normal blood glucose after 13 h which suggest a defect in ketolysis. In his cultured skin fibroblasts succinyl-CoA:acetoacetate transferase was deficient (residual activity 15%). Treatment in the acute phase consisted of sodium bicarbonate. At the present age of 9 years, psychomotor and physical development are within normal limits. CONCLUSION: Defects of ketolysis probably are underdiagnosed disorders and should be considered in infants and young children with persistent ketosis.

Acetoacetates↗

3-Methylisoxazol-5-one, an artefact from acetoacetic acid formed during urinary organic acid analysis.

During the analysis of urine for organic acids for suspected metabolic disorders by solvent extraction, derivatisation and capillary gas chromatography, unaccountably large lactic acid peaks were observed in some samples containing large amounts of acetoacetic acid. Electron impact mass spectrometry showed that this was due to two unknown compounds coeluting with lactic acid. These were found to be two trimethylsilyl derivatives of 3-methylisoxazol-5-one, produced from acetoacetic acid during oximation with hydroxylamine hydrochloride, by a cyclisation reaction. Awareness of the formation of this previously unreported artefact is important to laboratories employing a similar profiling procedure.

Acetoacetates↗

Arterial effects of palmitic, linoleic and acetoacetic acid.

The ultrastructural effects of a single brief intra-arterial infusion of palmitic, linoleic and acetoacetic acid on the arterial endothelium of the rat were investigated, and the following results obtained: (1) Palmitic acid, infused at a concentration of 4 mM/l, damaged the arterial lining by producing large cytoplasmic clefts and occasional blebbing and lysis of the endothelial cells. By contrast, linoleic acid, infused at the same concentration, had no damaging effects on arterial endothelium. (2) Acetoacetic acid damaged the arterial wall when infused at concentrations of 0.2 mM/l or higher by inducing extreme swelling and loss of cristae of the mitochondria in arterial endothelium and myocytes. The above results raise the possibility that (a) high saturated fatty acid diets may promote atherosclerosis not only by inducing hypercholesterolemia but also by injuring the arterial lining, and (b) diabetes may promote atherosclerosis not only by inducing hyperlipemia but also by damaging the arterial wall during periods of uncontrolled ketoacidosis.

Acetoacetates↗

In vivo utilization of [3-14C]acetoacetate for lipid and amino acid synthesis in the 15-day-old chick.

1. The utilization of different concentrations of acetoacetate for the in vivo synthesis of lipids and amino acids has been studied in brain, spinal cord, liver, small intestine and kidney of 15-day-old chicks. Both lipid and amino acid synthesis increased in an almost linear fashion with precursor concentration in the five tissues mentioned. 2. Lipid synthesis was very high in spinal cord, followed in decreasing order by brain, small intestine, liver and kidney. At the highest concentration assayed (48 mM) the main lipids synthesized were triglycerides in liver (75%) and kidney (52%) and cholesterol in brain (47%), spinal cord (47%) and small intestine (42%). 3. Amino acid synthesis from acetoacetate did not vary markedly among the five organs, although brain and spinal cord showed higher rates at the maximal concentrations of precursor. Glutamate was always the main amino acid formed.

Acetoacetates↗

Kinetic study of the reaction of acetoacetate with glycine and sodium nitroprusside.

This paper describes an extensive kinetic study of the reactions involved in the determination of acetoacetate in body fluids. It is concluded that acetoacetate reacts with glycine to produce an imine intermediate that tautomerizes to an enamine. It is also concluded that nitroprusside reacts with the imine intermediate to produce an unstable product with an absorption maximum near 540 nm. This product decays slowly to produce a stable product with an absorption maximum near 393 nm. A proposed reaction pathway is used to develop kinetic equations, rate constants, equilibrium constants, and molar absorptivity of the unstable product that permit quantitative prediction of the kinetic behavior for a wide range of reactant concentrations.

Acetoacetates↗

Synthesis and characterization of polyester dendrimers from acetoacetate and acrylate.

New aliphatic polyester-type dendrimers were synthesized using a new AB2-type building block 3, prepared from benzyl acetoacetate and 2 equiv of tert-butyl acrylate by acetoacetic acid ester synthesis. The reiterative [deprotection by HCO2H, then EDCI/DMAP coupling] sequence using divergent growth method gave [G1]-4tBu-[G5]-64tBu dendrimers. 13C NMR relaxation time (T1) measurements on the carboxy carbons show that the extended chain conformations are predominant in CDCl3. [structure: see text]

Acetoacetates↗

The effect of acetoacetate on plasma insulin concentration.

1. Sodium acetoacetate was infused into the inferior vena cava of fed rats, 48h-starved rats, and fed streptozotocin-diabetic rats treated with insulin. Arterial blood was obtained from a femoral artery catheter. 2. Acetoacetate infusion caused a fall in blood glucose concentration in fed rats from 6.16 to 5.11mm in 1h, whereas no change occurred in starved or fed-diabetic rats. 3. Plasma free fatty acids decreased within 10min, from 0.82 to 0.64mequiv./l in fed rats, 1.16 to 0.79mequiv./l in starved rats and 0.83 to 0.65mequiv./l in fed-diabetic rats. 4. At 10min the plasma concentration rose from 20 to 49.9muunits/ml in fed unanaesthetized rats and from 6.4 to 18.5muunits/ml in starved rats. There was no change in insulin concentration in the diabetic rats. 5. Nembutal-anaesthetized fed rats had a more marked increase in plasma insulin concentration, from 30 to 101muunits/ml within 10min. 6. A fall in blood glucose concentration in fed rats and a decrease in free fatty acids in both fed and starved rats is to be expected as a consequence of the increase in plasma insulin. 7. The fall in the concentration of free fatty acids in diabetic rats may be due to a direct effect of ketone bodies on adipose tissue. A similar effect on free fatty acids could also be operative in normal fed or starved rats.

Acetoacetates↗

Glucose metabolism in perfused skeletal muscle. Effects of starvation, diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptake and disposition.

1. The regulation of glucose uptake and disposition in skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin and exercise, induced by sciatic-nerve stimulation, enhanced glucose uptake about tenfold in fed and starved rats, but were without effect in rats with diabetic ketoacidosis. 3. At rest, the oxidation of lactate (0.44 mumol/min per 30 g muscle in fed rats) was decreased by 75% in both starved and diabetic rats, whereas the release of alanine and lactate (0.41 and 1.35 mumol/min per 30 g respectively in the fed state) was increased. Glycolysis, defined as the sum of lactate+alanine release and lactate oxidation, was not decreased in either starvation or diabetes. 4. In all groups, exercise tripled O2 consumption (from approximately 8 to approximately 25 mumol/min per 30 g of muscle) and increased the release and oxidation of lactate five- to ten-fold. The differences in lactate release between fed, starved and diabetic rats observed at rest were no longer apparent; however, lactate oxidation was still several times greater in the fed group. 5. Perfusion of the hindquarter of a fed rat with palmitate, octanoate or acetoacetate did not alter glucose uptake or lactate release in either resting or exercising muslce; however, lactate oxidation was significantly inhibited by acetoacetate, which also increased the intracellular concentration of acetyl-CoA. 6. The data suggest that neither that neither glycolysis nor the capacity for glucose transport are inhbitied in the perfused hindquarter during starvation or perfusion with fatty acids or ketone bodies. On the other hand, lactate oxidation is inhibited, suggesting diminished activity of pyruvate dehydrogenase. 7. Differences in the regulation of glucose metabolism in heart and skeletal muscle and the role of the glucose/fatty acid cycle in each tissue are discussed.

Acetoacetates↗

Activities of enzymes of acetoacetate metabolism in rat brown adipose tissue during development.

The activities of two mitochondrial enzymes concerned in the utilization of acetoacetate, namely 3-oxoacid CoA-transferase and acetoacetyl-CoA thiolase, were high throughout the suckling and weanling period in brown adipose tissue of the rat. In contrast, 3-hydroxybutyrate dehydrogenase activity was comparatively low during this period. The activity of cytosolic acetoacetyl-CoA synthetase (involved in lipogenesis) declined after birth and remained low until the pups were weaned. Experiments with brown-adipose-tissue slices from weanling rats indicated that 70% of the [3-14C]acetoacetate utilized was oxidized to 14CO2, and this value was not altered appreciably by the addition of glucose and insulin.

Acetoacetates↗

Propionate enhancement of acetoacetate oxidation in perfused rat hearts.

1. Isolated perfused rat hearts, acetoacetate oxidation was significantly enhanced by propionate. 2. No enhancement of acetoacetate oxidation could be observed in vitamin B12-deficient rat hearts when propionate was present. 3. Although the mechanisms of enhancement are not clear, ketone clearance is enhanced by propionate and may provide an insight into the therapy of certain diseases characterized by ketosis.

Acetoacetates↗

Hepatoprotective action of PGE1 analogue estimated by measuring the concentrations of acetoacetate and beta-hydroxybutyrate.

The research was carried out on 56 Chinchilla rabbits. The rabbits were divided into 8 groups with one control group. By means of stomach tube the rabbits were administered respectively: carbon tetrachloride (CCl4), synthetic analogue of prostaglandin misoprostol, CCl4 with misoprostol, indomethacin, misoprostol with indomethacin and CCl4 with misoprostol and indomethacin simultaneously. In order to confirm cytoprotective activity of prostaglandin E1 synthetic analogue in experimental acute hepatocellular damage, we measured in arterial blood the concentration of acetoacetate and beta-hydroxybutyrate and calculated molar ratio of their concentrations. The molar ratio of acetoacetate to beta-hydroxybutyrate which had been calculated for individual groups, regardless of the observed changes in ketone bodies' concentrations, in the case of rabbits which were administered CCl4 with misoprostol, had values similar to the ones obtained for the control group. The smallest value of the ketone body concentration ratio was stated at rabbits which were given only CCl4. The results of the research carried out unambiguously indicate hepatoprotective action of the synthetic analogue PGE1 which was administered to the rabbits.

3-Hydroxybutyric Acid↗

The association between calcium and acetoacetate, 3-hydroxybutyrate, pyruvate and lactate as determined by potentiometry. Determination of concentrational complexity constants in watery solutions at physiological conditions.

The complex binding of calcium to acetoacetate, 3-hydroxybutyrate, lactate and pyruvate was studied under physiological temperature and ionic strength by potentiometric measurement of the calcium ion activity. The limiting conductances of the complex-forming anions were measured to make a proper correction for the analytical error due to the liquid junction potential between the test solution and the salt bridge solution of the reference electrode. The calcium activity measurements indicate a 1:1 binding ratio of calcium and the anions. The concentrational complexity constants at physiological ionic strength of calcium and acetoacetate, 3-hydroxybutyrate, lactate and pyruvate are 8.8, 4.0, 11.6 and 6.3 1/mol, respectively, and the limiting conductances of the anions are 4.5, 3.8, and 5.2 X 10(-3) S.m2/mol, respectively. The thermodynamical complexity constants, which are based on ion activities, are about three times higher than the concentrational complexity constants. The possible role of the various complexes in lactic acidosis and ketoacidosis is discussed, and it is concluded that under merely moderate lactic acidosis the quantitatively most important of the low-molecular weight calcium complexes is the calcium-lactate complex.

3-Hydroxybutyric Acid↗

[Variations in the pattern of ruminal volatile free fatty acids, acetoacetate and glucose in the blood plasma of lactating cows after a period of fasting and renewed feeding].

3 lactating cows were fasted for 4 days and then again received food. The trial was performed to investigate the effect of fasting on the concentration and molar proportion of volatile fatty acids in the rumen, on the concentration of free fatty acids and glucose in blood plasma and on the acetoacetate concentration in the blood. Fasting produced a considerable decrease in the total concentration of volatile fatty acids. Processes of ruminal fermentation were characterized, during the fasting period, by a continuous increase in the molar proportions of acetic acid, isobutyric acid and isovaleric acid whereas a significant decline observed to occur in the molar proportions of propionic acid and butyric acid. The reciprocal behaviour of acetate and propionate under conditions of fasting lead to a linear increase in the C2 to C3 ratio from 4.4 : 1 to 9,6 : 1. With renewed food supply all fermentation data returned to normal levels. The concentrations of blood glucose declined after 24 hrs of fasting, remained at the same level for the following 2 days and rose again to normal values on the 4th day of fasting. The concentration of acetoacetate decreased significantly on the first day of fasting to values that were 4 times as high as the initial value. The concentration of free fatty acids in blood plasma increased tremendously to a value 40 times as high as that observed at the beginning of the fasting period. It proved to be a good indicator for the energy requirements of the organism.

Acetoacetates↗

Evaluation of Ketostix for plasma acetoacetate.

Acetoacetate in plasma is measured conveniently by Ketostix (Ames, Miles Laboratories, Elkhart, IN), a reagent strip test. The color formed on the reactive tip by the acetoacetate after 15 seconds is compared with the color chart provided for quantitation. When the concentration is between 4 to 12 mmol/L, the authors recommend a reassay using a diluted sample and both results averaged. Results thus determined agreed well with those obtained by a GC method. Analyst bias in color matching is not a serious problem. Ketostix stability and user performance are discussed in relation to quality control.

Acetoacetates↗

Nonequilibrium of two redox couplets in human plasma: lactate-pyruvate and beta-hydroxybutyrate-acetoacetate.

We examined the extracellular equilibrium status of two redox couplets normally found in plasma (lactate-pyruvate [L-P], beta-hydroxybutyrate, and acetoacetate) during acute metabolic acidosis produced by muscle exertion. Both pre- and postexertion plasma spontaneously underwent loss of acetoacetate and gain of L when compared to the baseline values. Exercise further induced a 332% rise in L (p less than .001) and a 102% rise in P (p less than .001). The empirically derived ratio of equilibrium constants, KLP/KBA, fell 50% (p less than .001), and the calculated change in free energy (delta F) fell from 3.6 to 3.1 kcal/mol (p less than .001) after exercise. The changes induced by exertion were simulated closely by an in vitro model of a reduced state. Thus, the triad of inconstant metabolite concentrations, inconstant KLP/KBA, and delta F both inconstant and non-zero, indicates that there is no state of equilibrium for these metabolite couplets in human plasma. The KLP/KBA ratio appeared to reflect the degree of deviation from equilibrium and may therefore be useful when investigating altered redox states such as metabolic acidosis.

3-Hydroxybutyric Acid↗