Beta-hydroxy-beta-methylbutyrate Research Articles

3-Hydroxy-3-methylglutaryl-CoA lyase deficiency as detected by radiochemical assay in cell extracts by thin-layer chromatography, and identification of three new cases

In this rapid radiochemical assay for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (I) activity in cell extracts, DL-3[glutaryl-3-14C]hydroxy-3-methylglutaryl-coenzyme A is used as substrate and the radiochemical product, [3-14C]acetoacetic acid, is converted to the more stable [3-14C]-3-hydroxybutyric acid in the presence of added NADH and 3-hydroxybutyrate dehydrogenase. Substrate and product are separated and quantified by thin-layer chromatography on cellulose (solvent system: butanol/water/formic acid, 77:13:10 by vol). All reagents for the assay are commercially available. No detailed column chromatography or spectrophotometry is required. Thus the assay is suited for any clinical laboratory. Using this procedure, we studied cultured fibroblasts or lymphocytes isolated from whole blood from five patients in whom the urinary organic acid profile was suggestive of deficiency of I. Three patients had less than or equal to 18% of control I activity in fibroblast or lymphocyte extracts. The other two had activity within the normal range. In one of the latter cases, urinary excretion of three of the characteristic acids disappeared with age, and 3-hydroxyisovaleric acid excretion was within normal limits. The other case presented with urinary excretion of moderate amounts of all four metabolites and the characteristic absence of urinary ketone bodies. Evidently, confirmatory enzyme studies should be undertaken, even when the profile of urinary organic acids appears definitive for this deficiency.

Quantification of urinary 3-hydroxyisovaleric acid using deuterated 3-hydroxyisovaleric acid as internal standard.

Deficiency of biotin at the tissue level can be assessed indirectly by measuring the urinary excretion of 3-hydroxyisovaleric acid. This paper describes the application of an improved method of quantifying urinary 3-hydroxyisovaleric acid using unlabeled and uniformly deuterated 3-hydroxyisovaleric acid. These compounds were synthesized by a modification of the lithioacetic acid method for generation of beta-hydroxy acids. Elemental analysis, nuclear magnetic resonance, gas chromatographic and gas chromatographic/mass spectrometric data demonstrated that the compounds are greater than 95% pure. Mass spectrometry confirmed the identity of the unlabeled compound, demonstrated that the deuterated compound is uniformly labeled, and offered insight into the pattern of mass fragmentation. The method for determination of the concentration of 3-hydroxyisovaleric acid in rat urine uses gas chromatographic/mass spectrometric quantification of the di-trimethylsilyl derivative with the deuterated compound as the internal standard. Results provide evidence that this method is more accurate than a previously published method that did not utilize the unlabeled and deuterated standards.

Physiological studies on the growth and utilization of sugars by Listeria species

Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency: review of 18 reported patients.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMG-CoA lyase) is an inborn error of leucine catabolism which often leads to life-threatening illness in the neonatal period. The cardinal clinical features include severe infantile hypoglycemia, metabolic acidosis, hepatomegaly, lethargy or coma and apnea. Hyperammonemia is variable. There is a characteristic absence of ketosis. Considerable heterogeneity has been observed in clinical and biochemical presentation. Acute episodes of illness have been mistaken for Reye syndrome. The pattern of organic acids in the urine includes large amounts of 3-hydroxy-3-methylglutaric, 3-methyl-glutaconic, 3-methylglutaric and 3-hydroxyisovaleric acids. Smaller, but appreciable levels of glutaric, adipic and other dicarboxylic acids may also be excreted in the urine. Lactic acid may be present in sizable amounts at times of acute illness. The primary defect is a deficiency of 3-hydroxy-3-methylglutaryl-coenzyme A lyase, a key enzyme in the cycle of ketogenesis.

Metabolism of leucine in fibroblasts from patients with deficiencies in each of the major catabolic enzymes: branched-chain ketoacid dehydrogenase, isovaleryl-CoA dehydrogenase, 3-methylcrotonyl-CoA carboxylase, 3-methylglutaconyl-CoA hydratase, and 3-hydroxy-3-methylglutaryl-CoA lyase.

The metabolism of leucine was studied in cultured human fibroblasts derived from patients with defects in each of the major steps in the catabolism of the amino acid. Intact fibroblasts were incubated with [U-14C]leucine and the organic acid products were isolated by liquid partition chromatography. In control fibroblasts the major product of leucine was 3-hydroxyisovaleric acid. This was also the case for fibroblasts with deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase, 3-methylcrotonyl-CoA carboxylase and 3-methylglutaconyl-CoA hydratase. There was little or no accumulation of the compound with fibroblasts from patients with maple syrup urine disease and isovaleric acidemia.

Hydroxy acid metabolites of branched-chain amino acids in amniotic fluid

A method is described in which ammonia chemical ionization gas chromatography-mass spectrometry was utilized in the selected ion monitoring mode to provide an accurate, selective approach to the quantification in amniotic fluid of a number of hydroxylated organic acids derived from the metabolism of the branched-chain amino acids. 2-Hydroxy- n-caproic acid was employed as an internal standard and the hydroxy acids were isolated from amniotic fluid by liquid partition chromatography and the trimethylsilyl derivatives were quantified. Normal values have been obtained for 2-hydroxyisovaleric acid, the sum of 2-hydroxyisocaproic acid and 2-hydroxy-3-methylvaleric acid, 2-methyl-3-hydroxybutyric acid, 3-hydroxyisovaleric acid and 2-ethyl-3-hydroxypropionic acid. The method also provides data on the concentration of methylmalonic acid. The concentration of 2-hydroxyisovaleric acid was not useful in the prenatal diagnosis of a fetus with maple syrup urine disease. Elevated concentrations of 2-methyl-3-hydroxybutyric acid as well as methylmalonic acid were found in the amniotic fluid of two fetuses with methylmalonic acidemia.

The identification and the excretion pattern of isovaleryl glucuronide in the urine of patients with isovaleric acidemia.

We identified isovaleryl glucuronide in the urine of patients with isovaleric acidemia by using gas chromatography-mass spectrometry (GC-MS) and by identifying the products of enzymatic hydrolysis. Conjugation of isovaleryl-CoA with glycine, by the action of glycine-N-acylase, is the main detoxification mechanism in isovaleric acidemia. The identification of isovaleryl glucuronide demonstrates a hitherto unknown, additional detoxification mechanism in patients with isovaleric acidemia. Quantitative analysis of 72 urine specimens from four patients with isovaleric acidemia shows that isovaleryl glucuronide is more likely to be excreted when the amount of urinary 3-hydroxyisovaleric acid excretion is high. This suggests that detoxification via glucuronide conjugation plays an important role when the glycine conjugation system is saturated.

Hydroxycarboxylic and oxocarboxylic acids in urine:products from branched-chain amino acid degradation and from ketogenesis

Hydroxy- and oxomonocarboxylic acids in urine of healthy individuals and of patients with diabetic ketoacidosis are analysed as methyl esters and methyl esters/O-methyloximes, respectively, by gas chromatography and gas chromatography-mass spectrometry. The derivatives are pre-fractionated by thin-layer chromatography. The acids originate mainly from ketogenesis and from the metabolism of valine, leucine and isoleucine. The amino acid metabolites fall into three groups: the 2-oxocarboxylic acids (2-oxoisovaleric acid, 2-oxoisocaproic acid and 2-oxo-3-methylvaleric acid); the 2-hydroxycarboxylic acids (2-hydroxyisovaleric acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylvaleric acid); and the 3-hydroxycarboxylic acids (3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, 3-hydroxy-2-ethylpropionic acid, threo-3-hydroxy-2- methylbutyric acid and erythro-3-hydroxy-2-methylbutyric acid). The threo form of 3- hydroxy-2-methylbutyric acid is the major constituent within the diastereomeric pair. Of the three groups of amino acid metabolites, the 3-hydroxycarboxylic acids in particular are elevated during ketoacidosis. The characteristic general features of the mass spectrometric fragmentation of the derivatives of the identified components are systematically described. The discussion of the fragmentation includes constituents of low concentrations, such as 3-oxocaproic acid, 4-oxo-butyric acid and 5-oxocaproic acid, which can be detected only when the pre-fractionation technique is applied.

N-Isovalerylalanine and N-isovalerylsarcosine: two new minor metabolites in isovaleric acidemia

Besides N-isovalerylglycine, isovaleric, 3-hydroxyisovaleric, 4-hydroxyisovaleric, methylsuccinic, mesaconic, 3-hydroxyisoheptanoic and N-isovalerylglutamic acids the excretion of hitherto unreported N-isovalerylalanine and N-isovalerylsarcosine, two minor but characteristic constituents of the organic acid profile in isovaleric acidemia, is described. The new metabolites are assumed to be formed from isovaleryl-CoA by action of the enzyme glycine N-acylase on alanine and sarcosine, respectively.

Organic acids or urine in multiple sclerosis.

The urinary excretion of organic acids was examined in 509 cases with multiple sclerosis and in 50 age- and sex-matched controls. The concentrations of the acids were related to creatinine. No differences were found for compounds such as glycolic acid, 2-methyl-3-hydroxybutyric acid, 2-ethylhydracrylic acid, 4-hydroxyphenylacetic acid, suberic acid and many other acids. However, the mean excretion of 2-hydroxy-2-methyl-3-butenoic acid was increased two-fold in the MS group. 2 MS cases had a very high excretion of 3-methylglutaconic acid, and another 6 cases had moderate elevations, which were fairly constant over a time period of several months. Moderate elevations were also noted in 2 healthy controls. 1 MS case had a very high excretion of 3-hydroxyisovaleric acid. 7 MS cases, and none in the control group, had elevated excretion of adipic acid. Differences were also noted for lactic acid, succinic acid, aconitic acid and 3-methyladipic acid. An oral dose of deuterium-labelled acetate was given to one of the patients with high excretion of 3-methylglutaconic acid. Deuterium was incorporated into this metabolite. 3-methylglutaconic acid, 2-hydroxy-2-methyl-3-butenoic acid, 3-hydroxy-isovaleric acid and 3-methyladipic acid are all potential isoprenoid metabolites. A possible defect in the pathway of isoprenoid biosynthesis is discussed.

Studies of urinary organic acid profiles of a patient with dihydrolipoyl dehydrogenase deficiency

Using gas chromatography-mass spectrometry (GC/MS), urinary organic acid profile studies were carried out on a patient with dihydrolipoyl dehydrogenase (E 3) deficiency. Elevated levels of 2-hydroxyglutaric acid, 2-hydroxyisocaproic acid and 2-oxoisocaproic acid were observed in addition to lactic acid, 2-oxoglutaric acid, 2-hydroxyisovaleric acid and 2-hydroxybutyric acid previously described in patients with E 3 deficiency. The 2-oxoglutaric acid levels were significantly lowered after branched-chain amino acid restriction. In an acute period, the patient was slightly ketoacidotic and excreted larger amounts of 2-oxoglutaric acid and lactic acid than in a static period. It was shown that, prior to confirmatory enzyme studies, patients with E 3 deficiency who were suspected to have atypical maple syrup urine disease or chronic lactic acidosis can be rapidly identified by GC/MS analysis of urinary acids.

Stable isotope dilution analysis of 3-hydroxyisovaleric acid in amniotic fluid: contribution to the prenatal diagnosis of inherited disorders of leucine catabolism.

A quantitative assay for 3-hydroxyisovaleric acid in amniotic fluid was developed using D6-3-hydroxyisovaleric acid as an internal standard. 3-Hydroxyisovaleric acid was isolated by liquid partition chromatography and the amount determined by selected ion monitoring, ammonia chemical ionization gas chromatography-mass spectrometry of the trimethylsilyl derivatives. The concentration of 3-hydroxyisovaleric acid in ten normal amniotic fluid was 4.52 +/- 1.73 mumol/l. The level was elevated eight-fold in the amniotic fluid from a pregnancy resulting in the birth of a child with biotin-responsive multiple carboxylase deficiency. The stable isotope dilution assay of 3-hydroxyisovaleric acid in amniotic fluid is a rapid, sensitive and accurate method for the prenatal diagnosis of this disorder, and may be of value in the prenatal diagnosis of other inherited disorders of leucine catabolism.

Formation of β-hydroxyisovalerate by an α-ketoisocaproate oxygenase in human liver

Previously we demonstrated the occurrence of a soluble dioxygenase in rat liver which converts α-ketoisocaproic acid (the keto acid analog of leucine) to β-hydroxyisovaleric acid. Herein we show that human liver contains a similar soluble enzyme which converts α-ketoisocaproate to β-hydroxisovaleric acid. We suggest this enzyme functions as a “safety valve” in liver to help prevent excessive accumulation of α-ketoisocaproate.

Incorporation of radioactive precursors into beauvericin produced by Paecilomyces fumoso-roseus

The biosynthesis of the depsipeptide antibiotic beauvericin was studied by feeding submerged cultures of Paecilomyces fumoso-roseus with [ 14C]-labelled precursors. l-Phenylalanine, hydroxyisovaleric acid, l-valine and methyl-labelled methionine were efficiently incorporated into beauvericin, whereas no radioactive depsipeptide was formed with l-leucine and l-tyrosine. [ 14C]Phenylalanine was found to label exclusively the N-methylphenylalanine residues of beauvericin, the N-methylgroups, thereof, were derived from methionine. Beauvericin synthesized in the presence of dl-[2- 14C]hydroxyisovaleric acid or l-[ 14C]valine was specifically labelled in its hydroxy valeryl moieties.

Inherited 3-methylglutaconic aciduria in two brothers—Another defect of leucine metabolism

Two brothers, aged 7 and 5 years, who excreted large amounts of the leucine metabolites 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid, are described. The excretion of these metabolites could be enhanced by increasing the leucine intake. Restriction of the protein intake resulted in a marked reduction of the metabolite excretion. However, the excretion of the ultimate leucine metabolite, 3-hydroxy-3-methylglutaric acid, remained unchanged at a low level. The only clinical abnormality was speech retardation. A (partial) deficiency of 3-methylglutaconyl coenzyme A hydratase is proposed to be the most likely underlying defect.

Increased excretion of lactate, glutarate, 3-hydroxyisovalerate and 3-methylglutaconate during clinical episodes of propionic acidemia

Metabolic changes dependent upon clinical conditions were studied in an eightmonth-old girl with propionyl CoA carboxylase deficiency. Only methylcitric acid and 2-methyl-3-oxovaleric acid were detected in the urine of the patient under clinically favorable conditions. During episodes of clinical decompensation, she excreted increased amounts of all the metabolites associated with this disorder. Four acetyl CoA precursors increased during clinical episodes: glutaric acid, a catabolic intermediate of lysine; 3-hydroxyisovaleric acid and 3-methylglutaconic acid, catabolic intermediates of leucine; and lactic acid. This suggests that under clinically favorable conditions the patient has an altered propionate metabolism which proceeds via normal acetyl CoA metabolism with sufficient capacity for acetyl CoA plus propionyl CoA metabolism. When the production of propionyl CoA exceeds the metabolic capacity, however, the catabolism of potent ketogenic amino acids is effectively suppressed in order to reduce acetyl CoA production.

Gas-chromatographic/mass spectrometric detection of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency in double first cousins.

Gas chromatography/mass spectrometry was used for the detection of 3-hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) deficiency in double first cousins. This enzyme is in the last step of leucine catabolism and is also involved in ketogenesis. Quantitation of urinary organic acids as their cyclohexyl esters demonstrated increased concentrations of 3-hydroxy-3-methylglutaric acid, 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid. The procedure is more rapid, sensitive, and specific than previously reported gas-chromatographic methods for acid quantitation. The affected children initially presented with symptoms similar to Reye's syndrome; the acids were quantitated during periods of altered intake of protein and fat. Both leucine and fat intake contributed to increased acid excretion. These studies suggest that life-threatening episodes of hypoglycemia are best prevented with a low-protein, low-fat diet.

Clinical and Metabolic Abnormalities in a Boy with Dietary Deficiency of Biotin

Dietary deficiency of biotin was documented in an 11-year-old retarded boy as a consequence of a dietary prescription containing raw eggs. Clinical manifestations were alopecia totalis and an erythematous, exfoliative dermatosis. Metabolic characteristics included increased excretion of 3-methylcrotonylglycine, 3-hydroxyisovaleric acid, 3-hydroxypropionic acid, methylcitric acid, and lactic acid, as well as a propensity for the development of ketosis. The activities of propionyl coenzyme A carboxylase and 3-methylcrotonyl coenzyme A carboxylase in extracts of leukocytes were deficient. Treatment with biotin and the removal of raw eggs, which contain the biotin-binding protein, avidin, from the diet led to the reversal of all of the clinical and metabolic manifestations observed.

Biotin-responsive in vivo carboxylase deficiency in two siblings with secretory diarrhea receiving total parenteral nutrition

Two siblings with a congenital syndrome of secretory diarrhea and seizures developed progressive skin rash, alopecia, and mucocutaneous candidiasis while receiving biotin-free total parenteral nutrition. Abnormally low urinary biotin excretion was associated with these clinical findings, but the serum concentration of biotin was within the normal range. There was also increased urinary excretion of lactic acid, 3-hydroxyisovaleric acid, 3-hydroxypropionic acid, and 3-methylcrotonylglycine. The younger of the two children subsequently died with severe metabolic acidosis. In the oder sibling, intravenous treatment with biotin (200 micrograms/day) resulted in resolution of the organic aciduria. A larger dose (10 mg/day) appeared to be required for rapid improvement in the skin lesions. These cases suggest that clinically significant biotin deficiency can occur in patients with chronic diarrhea receiving biotin-free total parenteral nutrition.

4-hydroxyisovaleric acid: a new metabolite in isovaleric acidemia.

In addition to N-isovalerylglycin, isovaleric, 3-hydroxyisovaleric and methylsuccinic acids the excretion of previously unreported 4-hydroxyisovaleric acid in isovaleric acidemia is described. The new metabolite seems to be an intermediate product in the formation of methylsuccinic acid from isovaleric acid by omega-oxidation.