The urinary organic acid profile associated with 3-hydroxy-3-methylglutaric aciduria.

The urinary organic acid profile of 3-hydroxy-3-methylglutaric aciduria contains excessive amounts of 3-methylglutaconic acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxyisovaleric acid and 3-methylglutaric acid. The previously reported 3-methylcrotonic acid was shown to be an artefact and is not a diagnostic metabolite for this defect.     

3-Hydroxy-3-methylglutaric aciduria: 3-hydroxy-3-methylglutaryl-coenzyme A lyase levels in leucocytes.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase activity in leucocytes from a baby with 3-hydroxy-3-methylglutaric aciduria is markedly deficient when compared to controls. Both parents have reduced levels of the enzyme in leucocytes.     

Studies on a child suspected of having a dficiency in 3-hydroxy-3-methylglutaryl-Co A lyase

The urine of a child who presented with hyperammonemia was found to contain elevated levels of 3-hydroxy-3-methylglutaric acid, 3-methylglutaconic acid and 3-methylglutaric acid. An increased excretion of these organic acids has been reported previously in a child with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Enzyme studies using cultured fibroblasts from this patient, however, indicated that the 3-hydroxy-3-methylglutaryl-CoA lyase activity was not markedly reduced.     

Heterogeneity of erythrocyte pyruvate kinase deficiency and related metabolic disorders in patients with hematological diseases.

In several patients suffering from congenital non-spherocytic hemolytic anemia or from malignant hemotological disorder associated with erythrocyte pyruvate kinase (PK) deficiency, a metabolic study has been carried out involving the following biochemical determinations: assay of red cell enzyme activities; estimation of glucose consumption; measurement of the rate of glycolytic intermediates; and, in some cases, enzyme purification and characterization of the PK variant. Metabolic equilibrium most probably does not depend on kinetic characteristics of PK molecules. Furthermore, the data obtained allow separation of cases with congenital non-spherocytic hemolytic anemia (hereditary defect) and acquired PK deficiencies.     

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3, 17-dihydroxy-5β-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3, 11β-dihydroxy-5-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P₃) and 11-keto-pregnanetriol (11-keto-P₃). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3ξ, 16ξ, 17ξ, 20ξ-pregnanetetrol (16-OH-P₃) and surprisingly also a 21-hydroxylated compound, namely 3β, 20, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency.

The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of .the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.     

3-Methylcrotonylglycine excretion in 3-hydroxy-3-methylglutaric aciduria

1. 3-Methylcrotonylglycine was identified in urine from an infant with 3-hydroxy-3-methylglutaric aciduria. 2. The concentration of 3-methylcrotonylglycine in urine was approximately one sixth of that of the other metabolite of 3-methylcrotonyl-CoA, 3-hydroxyisovaleric acid. 3. The presence of both metabolites in the infant's urine indicates an inhibition of 3-methylcrotonyl-CoA carboxylase activity in tissues of the infant.     

The absolute configuration of urinary 2-hydroxybutyric acid in patients with ketosis and lactic acidosis

The absolute configurations of urinary 2-hydroxybutyrate and 3-hydroxybutyrate were determined in patients with lactic acidemia and ketosis by capillary gas-liquid chromatography of their O-acetylated (?)-menthyl ester derivatives. 2-Hydroxybutyrate had the L-configuration, whereas 3-hydroxybutyrate was in the d-configuration.     

A patient with purine nucleoside phosphorylase deficiency: enzymological and metabolic aspects.

1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. Incubation of intact NP-deficient red cells with [14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. Incubation with [14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. Incubation with [14C]hypoxanthine and [14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples.     

Elevated levels of urinary acid maltase and maltose: Riboflavin glucosyltransferase activities in diabetic patients

Urinary acid maltase and maltose: riboflavin glucosyltransferase activities, assayed by measuring the enzymatic hydrolysis of maltose and the enzymatic formation of 5'-d-riboflavin-α-d-glucopyranoside from maltose, respectively, were significantly elevated in urine of diabetic patients.     

2-Ethylhydracrylic acid: a newly described urinary organic acid

2-Ethylhydracrylic acid has been identified as a significant component in the organic acid fraction of normal human urine. Its excretion is increased after an isoleucine load in a normal subject and was elevated in the urine of a child with an apparent inborn error of metabolism of isoleucine at the 3-ketoacyl thiolase step.     

Urinary amino acid excretion by patients with β-thalassemia

Twenty-four hour urinary amino acid excretion has been studied in patients with β-thalassemia (four adult major, two adult intermedia, and three children major). β-Aminoisobutyric acid was found to be increased (14-fold on the average) in patients with β-thalassemia without evidence for increase in the excretion of most other amino acids. No correlation of β-aminoisobutyric acid excretion with the hematological status of the patient was found. Elevated taurine excretion (2-fold) was also noted in the adult patients with β-thalassemia. Greatly increased excretion of β-aminoisobutyric acid appears to correlate with a poor prognosis and may reflect generalized tissue catabolism rather than being a specific indicator of ineffective erythropoiesis.     

Abnormalities in folate binding pattern of serum from a patient with megaloblastic anemia and folate deficiency

Effects of various experimental conditions on serotonin (5-HT) uptake in human platelets were examined. The experimental design allowed the evaluation of the effect of diffusion and other non-saturable processes on the affinity and maximum activity of the membrane pump for 5-HT uptake. Total 5-HT uptake was determined by incubating platelet-rich plasma (PRP) with increasing concentrations of serotonin at 37°C for 4 min. The passive uptake was measured by the addition of various 5-HT concentrations to PRP in buffer at 37°C, followed by immediate transfer to an ice-cold water bath. The difference between the total and passive uptake was linear for 6 min. The affinity (K_m) for active platelet serotonin uptake was 0.45 ± 0.09 γmol/l and maximal rate of uptake (V) was 10.7 ± 2.1 pmol/10⁷ platelets/min. The described method provides a convenient and reliable measure of active 5-HT uptake suitable for clinical investigation. The effect of passive diffusion on kinetic parameters is discussed.     

The urinary excretion of orotic acid and orotidine, measured by an isotope dilution assay

Unknown concentrations of orotic acid can be measured by competition with a known amount of [carboxyl-¹⁴C] orotic acid for reaction with a limiting amount of phosphoribosylpyrophosphate in the presence of orotate phosphoribosyltransferase and orotidine monophosphate decarboxylase. The dilution of the specific radioactivity in the product ¹⁴CO₂ is a sensitive and accurate measure of the amount of orotic acid present in the sample. Orotidine can also be determined after hydrolytic cleavage to orotic acid.

The method was used to measure orotic acid and orotidine in urine samples from newborns, healthy controls and patients with gout or deficiency of hypoxanthine-guanine phosphoribosyltransferase receiving allopurinol. Urinary excretion of orotic acid and orotidine in newborns was similar whether the infants were breast-fed or received milk powder. The excretion of orotidine was increased in all patients receiving allopurinol. After allopurinol administration orotic acid excretion was increased in gouty patients but close to normal values in patients with deficiency of hypoxanthine-guanine phosphoribosyltransferase. The results are discussed in relation to the mechanism by which allopurinol inhibits pyrimidine metabolism.     

Glycogenosis type II: the infantile- and late-onset acid maltase deficiency observed in one family.

The concurrence of the infantile and adult form of acid maltase deficiency in one family is presented. The muscle of both cases possesses residual activity. The glycogen amount in the infantile form is increased tremendously, while in the late-onset the amount of glycogen is normal.     

Analysis of glucocorticoid metabolites in the neonatal period: catabolism of cortisone acetate by an infant with 21-hydroxylase deficiency.

The urinary excretion of 27 individual 17-hydroxycorticosteroids by a female infant with 21-hydroxylase deficiency was studied using gas chromatography on an open-tubular column and combined gas chromatography-mass spectrometry. Samples were analysed from days 1, 3 and 4 of life, before cortisone acetate treatment commenced on day 6, and on days 9 and 192, during treatment. The pattern of metabolites before and after treatment showed similarities with extensive reductions at positions 3 and 20 of the steroid nucleus and hydroxylations at positions 1 and 6. The following new glucocorticoid metabolites were identified: 1beta,3alpha,17alpha,21-tetrahydroxy-5alpha(andbeta)-pregnane-11,20-dione; 3alpha,6xi,17alpha,21-tetrahydroxy-5xi-pregnane-11,20-dione (2 epimers); 1beta,3alpha,17alpha,20alpha(andbeta),21-pentahydroxy-5beta-pregnan-11-one, and 3alpha,6xi,17alpha,20alpha (andbeta),21-pentahydroxy-5beta-pregnan-11-one. These steroids comprised 50% of the total metabolites on day 9, but only 20% on day 192. They have also been detected in the urine of normal neonates.     

The role of common urinary constituents in the precipitation of ammonium acid urate

A high proportion of the inhibitory activity shown by urine toward precipitation of ammonium acid urate is ultrafilterable and most of this can be accounted for by the common, low molecular weight components of urine. The individual inhibitory effects of sodium chloride, sodium sulphate, potassium chloride, calcium chloride, magnesium sulphate, sodium dihydrogen phosphate, sodium pyrophosphate, citric acid, hippuric acid, creatinine and urea upon the precipitation of ammonium acid urate have been quantified in an aqueous test system.     

(2-Ethoxyethoxy)acetic acid: An unusual compound found in the gas chromatographic analysis of urinary organic acids

An unknown acidic compound was detected in a number of urine samples from patients with a suspected metabolic disorder and some patients treated with chemotherapy. The structure of this compound has been characterized as (2-ethoxyethoxy)acetic acid, using a gas chromatography/mass spectrometry/ computer system.The authentic compound was synthesized and compared with the unknown. Urinary (2-ethoxyethoxy)acetic acid is assumed to be formed endogenously from an exogenous precursor, probably 2-(2-ethoxyethoxy)ethanol.     

Unusual pattern of metabolites in the urine of child with tyrosinemia: glyceraldehyde

An unusual metabolite has been found in the acidic fraction of the urine of a child with tyrosinemia. This compound has been identified as glyceraldehyde, in addition to phenylacetic acid and p-hydroxybenzaldehyde, on the basis of gas chromatograph-mass spectral data and comparison with authentic samples.     

Endogenous levels of free and conjugated urinary 3-methoxy-4-hydroxyphenylethyleneglycol in control subjects and patients with pheochromocytoma determined by reversed-phase liquid chromatography with electrochemical detection

A sensitive and specific reversed-phase liquid-chromatography (HPLC) method for the determination of urinary free and conjugated 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) was developed. Sample preparation is minimal and the method is ideally suited for routine clinical assays of this catecholamine metabolite.Reported in this paper are excretion profiles of control subjects and patients with pheochromocytoma. Quantitative results were expressed as μg MHPG per mg creatinine, in order to eliminate variations associated with the 24-h urine collection. The levels of free and conjugated MHPG, determined in 20 control subjects ranged between 0.04–0.11 μg/mg of creatinine and 0.48–1.25 μg/mg of creatinine, respectively. The corresponding levels of free and conjugated MHPG in 8 patients with pheochromocytoma were 0.29–1.21μg/mg of creatinine and 2.47–15.98 μg/mg of creatinine, respectively.The described liquid-chromatographic analysis, coupled with the highly sensitive electrochemical detection, provides a simple and reliable method for metabolic profiling of catecholamine by-products at their endogenous levels. This offers an attractive possibility for the detection of neural crest lesions which may cause problems in diagnosis because of their small size, infrequent occurrence and symptoms similar to those of essential hypertension.     

Urinary excretion of orotic acid, orotidine and other pyrimidines in a patient with purine nucleoside phosphorylase deficiency.

Urinary orotidine and orotic acid have been determined in a patient with purine nucleoside phosphorylase (PNP) deficiency under various dietary therapeutic conditions. For this purpose a new procedure for the analysis of both compounds has been developed, consisting of prefractionation with Dowex 1X8, followed by two HPLC steps on a micro Bondapak NH2 and a micro Bondapak C18 column. With this method normal as well as slightly elevated excretions of orotic acid have been found in our patient. No evidence was obtained for inhibition of OPRT by purine (deoxy)nucleosides as a cause of pyrimidine starvation. A significant increase of urinary orotidine was found after loading with allopurinol. For comparison excretory values in a patient with ornithine transcarbamylase deficiency and also in a patient with orotic aciduria type I are shown. The possible cause of the slight increase in urinary orotic acid in our patient has been discussed.