Fa Hommes

(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.

Nonketotic hyperglycinemia: an in vitro study of the glycine-serine conversion in liver of three patients and the effect of dietary methionine.

Extract: Liver biopsies were performed on three patients suffering from the nonketotic form of hyperglycinemia. Direct assay of the enzymes involved in glycine-serine conversion revealed that the enzymic defect underlying this disease was due to nonfunction or absence of a glycine cleavage system. This enzyme system catalyzes the conversion of glycine to hydroxymethyltetrahydrofolic acid, carbon dioxide, and ammonia. In patients with hyperglycinemia, serine to glycine conversion is normal and patients depend on this metabolic conversion for their supply of C1 units. As glycine formed in this reaction cannot participate as a C1-unit donor, it cannot be metabolized further and consequently will accumulate.A therapeutic approach was devised to supply the organism with sufficient C1 units and was achieved by addition of methionine to a diet devoid of serine and glycine.As a consequence, the patient with hyperglycinemia does not rely so heavily on the glycine-serine conversion to fulfill his requirements for C1 units and glycine will not accumulate. Levels of glycine in plasma fell to normal values after feeding a diet with 0.3 g methionine/kg/24 h for a period of 10 days. It was demonstrated that the decrease in plasma glycine by administration of methionine was not due to increased excretion of glycine in the urine.Speculation: These studies have yielded evidence that the nonketotic form of hyperglycinemia is due to absence or nonfunction of the enzymic system responsible for the conversion of glycine to CO2, NH3, and hydroxymethyltetrahydrofolic acid. To meet the requirements of the organism for C1 units the conversion of serine to glycine proceeds at an accelerated rate. This can be compensated by addition of methionine to the diet, which serves as an alternate C1-unit donor. Patients with hyperglycinemia that do not respond to serine- and glycine-free diets may respond to diets rich in methionine.

Citrullinemia, Report of a Case, with Studies on Antenatal Diagnosis

Extract: A case is described of an infant with a deficiency of liver argininosuccinate synthetase, characterized by rapidly increasing drowsiness, respiratory insufficiency, and convulsions starting 67 hr after birth, which resulted in the death of the patient on the 4th day.Quantitative analyses of amino acids in blood plasma showed increased concentrations of citrulline (2.64 mM) lysine (1.25 mM), histidine (0.34 mM), glutamine (4.17 mM), proline (1.67 mM), glycine (1.35 mM), alanine (3.96 mM), and methionine (0.24 mM).The activity of argininosuccinate synthetase in a liver biopsy was found to be below the limits of detection, whereas the activities of carbamylphosphate synthetase and ornithine transcarbamylase (21.8 and 55.6 μmol/min × g wet wt, respectively) I were higher than those of the control tissue.The activity of arginase (141.1 μmol/min × g wet wt), an enzyme localized behind the enzymic block, was found to be lower than that of the controls (230–800 μmol/min × g wet wt).It is suggested that these differences are due to adaptation of urea cycle enzymes to protein intake as demonstrated by Nuzum and Snodgrass [14] in primates.The activity of argininosuccinate synthetase of a postmortem brain biopsy was found to be normal. This points to a different genetic origin (isoenzyme) of the brain enzyme.Cultured amniotic cells of a fetus from a pregnancy in the same family, taken in the 20th week of pregnancy, demonstrated incorporation of ureido-14C-citrulline into trichloroacetic acid (TCA)-soluble material to an extent half of that observed with cultured amniotic cells from a normal fetus (31 dpm/μg DNA and 68 dpm/μg DNA, respectively). This has been interpreted as being due to the heterozygotic state of the fetus.After a normal pregnancy a healthy boy was born.Speculation: The metabolic basis for neurologic abnormalities in citrullinemia is not known. In the present case a virtual absence of argininosuccinate synthetase of liver was found, whereas the activity of the brain enzyme was normal. As the blood ammonia concentration was never found to be abnormal, it is postulated that citrulline itself is toxic, i.e., inhibitory to a biochemical reaction.

Carbamylphosphate synthetase deficiency in an infant with severe cerebral damage.

The biosynthesis of urea from ammonia and carbon dioxide requires five enzymes. One enzyme, carbamylphosphate synthetase converts ammonia and bicarbonate to carbamyl phosphate, which subsequently condenses with ornithine to form citrulline, a reaction catalysed by ornithine transcarbamylase. Citrulline is then converted by argininosuccinic acid synthetase to argininosuccinic acid, which is cleaved to form arginine and fumaric acid. The enzyme arginase finally converts arginine to ornithine and urea. All of these enzymes, with the exception of arginase, have been associated with an inborn error of metabolism: hyperammonaemia with a defective ornithine transcarbamylase (Russell et al., 1962; Levin and Russell, 1967), citrullinaemia with a defective argininosuccinate synthetase (McMurray et al., 1962), and argininosuccinic aciduria with a defective argininosuccinic acid cleavage enzyme (Allen et al., 1958). Mental retardation is a common feature of these enzymatic defects, but it is uncertain whether the cerebral defect is due to accumulation of ammonia. There is increasing evidence that the urea cycle proceeds in the brain cells (Sporn et al., 1959; Kemp and Woodbury, 1965), so that brain damage may be due to toxic effects of accumulated urea cycle intermediates and not necessarily to a high ammonia level. Five cases with a high blood ammonia level (postprandial ammonaemia) due to enzymatic defects have been described. In 4 of these cases enzyme activity determinations have been carried out (Freeman et al., 1964; Russell et al., 1962; Levin and Russell, 1967). In the case described by Freeman et al. (1964) a reduced activity of carbamylphosphate synthetase with normal activities of the other enzymes of the urea cycle was found. The other 3 cases showed a very low activity of liver ornithine transcarbamylase, though the activity of carbamylphosphate synthetase was also below the normal value (see Addendum).

A rapid and sensitive method for the determination of short chain fatty acids in serum

Abstract A gas Chromatographie method is described for the rapid detection of elevated amounts of short chain fatty acids in small samples of serum. About 2 μl of acidified serum is brought directly onto the column. The temperature-programmed gas Chromatographie system permits a good and reproducible separation of the short chain fatty acids. The procedure takes about 45 min. Interference of other compounds normally occurring in serum could not be observed. Analysis of hydrolyzed urine (by the same gas Chromatographie procedure) may be applied to determine the excretion of short chain fatty acids during periods of remission. The small amount of serum needed and the speed of the method makes this method especially useful for the diagnosis of acidemias and acidurias, due to overproduction of volatile, short chain fatty acids in newborns.

DEVELOPMENT OF CREATINE-KINASE IN RAT SKELETAL-MUSCLE - CHANGES IN ISOENZYME RATIO, PROTEIN, RNA AND DNA DURING DEVELOPMENT

The activity of cytosolic creatine kinase in rat skeletal muscle rises stepwise during development. The increases occur simultnaeously with transient increases in DNA content. The second increase is accompanied by a rise in total protein, soluble sarcoplasmic protein and RNA/DNA ratio. Such changes are not observed at 20 days after birth, when creatine kinase finally accumulates to the adult level. Transient higher amounts of the MB and BB isoenzymes are observed after the first and second stepwise increase. The increase in creatine kinase activity observed after birth is predominantly due to an activation of the M gene. The BB isoenzyme is still present in adult skeletal muscle, but contributes little to the total activity.

EFFECT OF THIAMINE TREATMENT ON ACTIVITY OF PYRUVATE DEHYDROGENASE - RELATION TO TREATMENT OF LEIGHS ENCEPHALOMYELOPATHY

Extract: Rats received intraperitoneal injection of thiamine (125 mg/kg body wt) for 4 consecutive days. There was less inhibition by ATP (9% ± 2%) of the pyruvate dehydrogenase complex of isolated liver mitochondria from treated rats than in liver mitochondria from control rats (30% ± 8%). The liver mitochondria of the treated animals contained about 25% more thiamine pyrophosphate than the mitochondria of the controls (0.48 ± 0.03 and 0.36 ± 0.01 nmoles/mg mitochondrial protein, respectively).Roche and Reed have demonstrated inhibition of the phosphorylation of the pyruvate dehydrogenase complex by thiamine pyrophosphate, thereby maintaining pyruvate dehydrogenase in the active form. It is suggested that the therapeutic effect of high doses of thiamine given to patients who suffer from Leighs disease is, at least in part, due to maintainance of the pyruvate dehydrogenase complex in its active form, thus facilitating the oxidation of pyruvate.Speculation: Administration of high doses of thiamine to patients suffering from subacute necrotizing encephalomyelopathy seems to have a beneficial effect. Adverse effects, because of interference with a physiologic control mechanism, i.e., inhibition of the phosphorylation of the pyruvate dehydrogenase complex, may, however, interfere with its therapeutic value.

INDUCTION OF DT-DIAPHORASE BY BENZO(A)PYRENE IN RAT-LIVER - RELATION TO THE CELL-CYCLE

Abstract The kinetics of induction of cytosolic DT-diaphorase (NAD(P)H dehydrogenase-quinone, EC 1.6.99.2) by benzo(a)pyrene (BP) in the liver of the 8-day-old rat has been studied. After a lag phase of 8 h, DT-diaphorase reaches its maximum activity in three waves, with plateau levels of activity between 15–18, 26–36, and 40 h after administration of BP, at 4, 15, and 26 times the basal activity, respectively. A lower degree of induction of DT-diaphorase could be observed in the kidney cortex of the young rat and in the liver of the adult rat. No induction was observed in the fetal liver and in the adult kidney cortex. Lead acetate treatment of the adult rat resulted in induction of DT-diaphorase by BP in the liver and in the kidney cortex. Induction could not be observed in the regenerating liver of the adult rat. Experiments with picolinic acid (PA)—as a G1 inhibitor—administered simultaneously or at different time intervals after BP administration resulted in an inhibition of induction, depending on the time of administration of picolinic acid. It is concluded that a mitotic cell cycle is necessary for DT-diaphorase induction by BP. Evidence is presented that BP acts in late G1. The kinetics of induction of aryl hydrocarbon hydroxylase (AHH) by BP in liver microsomes of the 8-day-old rat has been compared with the induction of cytosolic DT-diaphorase. The effect of PA on the induction of AHH has also been studied. In view of the differences in kinetics of induction and in the effects of PA, it is concluded that the induction of AHH and that of DT-diaphorase are dissociated. AHH induction may take place in all hepatocytes, in contrast to DT-diaphorase induction.

The development of DT-diaphorase in rat liver and its induction by benzo(a)pyrene.

The development of cytosolic DT-diaphorase--NAD(P)H dehydrogenase, quinone, EC 1.6.99.2--and its induction by benzo(a)pyrene has been studied in rat liver. DT-diaphorase belongs to the late suckling cluster, because the largest increase in activity can be observed 18 days after birth. A considerable activity is present, however, in the neonatal period. The activity of the enzyme can be prematurely induced by benzo(a)pyrene. A lag phase of 10 h can be observed before the activity of DT-diaphorase starts to increase. This increase in activity proved to be sensitive to inhibitors of mixed-function oxydase and RNA and DNA synthesis.

A CASE OF METHYLMALONIC AND PROPIONIC ACIDEMIA DUE TO METHYLMALONYL-CoA CARBONYLMUTASE APOENZYME DEFICIENCY

Abstract. A patient presenting with a deep metabolic acidosis after birth is described. Gas chromatographic analysis of short chain fatty acid and non volatile organic acids revealed the presence of both propionic and methylmalonic acid. In plasma obtained immediately after death the propionic‐and methylmalonic acid concentrations were measured after separation of both acids by thin layer chromatography. The propionic acid concentration was about 5 mM while the methylmalonic acid concentration was 2.6 mM. The methylmalonic acid concentration in urine was 6.8 mM. Propionyl‐CoA carboxylase activity measured in leucocytes and liver‐mitochondria revealed normal values (53 pmoles/min/mg protein and 6.5 nmoles/min/mg protein respectively). 2‐ 14C‐Methyl‐rnalonate oxydation in intact fibroblasts was totally blocked in the patients cells. The methylmalonyl‐CoA carbonyl mutase activity was found to be absent in the patients fibroblasts. Addition of vit. B12 coenzyme to the incubation mixture stimulated 14C‐succinate formation in the control cells but not in the patients cells.