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The possible role of superoxide anion in 2-oxoglutarate-coupled dioxygenase reactions has been investigated. γ-Butyrobetaine hydroxylase (EC 1.14.11.1) was inhibited by human erythrocyte superoxide dismutase (EC 1.15.1.1), probably due to release of Cu2+ or Zn2+, as the inhibition was more pronounced after heat-inactivation of the dismutase and as Cu2+ was a potent inhibitor. Bovine superoxide dismutase and the Mn2+-containing superoxide dismutase from Escherichia coli were not inhibitory. Superoxide anion generated from xanthine/xanthine oxidase was not stimulatory and could not replace ascorbate. Thymine 7-hydroxylase (EC 1.14.11.6) and thymidine 2′-hydroxylase (EC 1.14.11.3) were not inhibited by erythrocyte superoxide dismutase or stimulated by superoxide anion. γ-Butyrobetaine hydroxylase was inhibited by a number of low-molecular-weight compounds, such as tetranitromethane, Nitro Blue Tetrazolium, adrenaline and Tiron, which may act as scavengers of superoxide anion. Involvement of this radical in other oxygenase reactions has been inferred from the findings that they were inhibitory for the respective enzymes. Several of these compounds also inhibited γ-butyrobetaine hydroxylase. It could be concluded from these experiments, however, that mechanisms other than disposal of superoxide anion might equally well be operative, such as hydrophobic interaction with the enzyme protein and interaction with compounds required for full enzymic activity, e.g. iron and ascorbate. The results appear to rule out a requirement for superoxide anion generated in free solution, and have not yielded evidence for participation of enzyme-bound superoxide anion in 2-oxoglutarate-dependent hydroxylations.

We report the finding of mitochondrial ATP-synthase deficiency in a child with persistent 3-methylglutaconic aciduria. The child presented in the neonatal period with severe lactic acidosis, which was controlled by Na-HCO3and glucose infusions. During the 1st y of life, there were several episodes of lactic acidosis precipitated by infections or prolonged intervals between meals. The excretion of lactate in urine was variable, but there was a persistent high excretion of 3-methylglutaconic acid. The activity of 3-methylglutaconyl-CoA hydratase in fibroblasts was normal. The child had a hypertrophic cardiomyopathy and magnetic resonance images revealed hypoplasia of corpus callosum. The gross motor and mental development was retarded, but there were no other neurologic signs. Investigation of muscle mitochondrial function at 1 y of age revealed a severe mitochondrial ATP-synthase deficiency (oligomycin-sensitive, dinitrophenol-stimulated Mg2+ATPase activity: 27 nmol x min-1x (mg protein)-1, control range 223–673 nmol x min-1x (mg protein)-1. The mitochondrial respiratory rate was low and tightly coupled. The respiratory rate was normalized by the addition of an uncoupler. Low Mg2+ATPase activity was also demonstrated by histochemical methods. Morphologic examination revealed ultrastructural abnormalities of mitochondria. There was no deletion of mitochondrial DNA. The sequences of the ATP synthase subunit genes of mitochondrial DNA were in accordance with published normal sequences.

Three cases of thallium poisoning occurred within the same family after eating a meal of poisoned sour milk. The 58 year old mother died after four days cardiovascular insufficiency. Massive thallium concentrations were found in blood, urine, liver and kidney. The 59 year old father presented after four days with a neurology typical for classic thallotoxicosis with potentially lethal blood thallium levels. This patient recovered after therapy including Prussian Blue, forced diuresis and two six hour hemoperfusions with a charcoal column. Urinary elimination of thallium over two weeks was almost 300 mg, whereas a maximum total of 63 mg were removed with hemoperfusion. A retrospective estimate of the total dose ingested and the large distribution volume of thallium suggest that hemoperfusion did not affect the course of the intoxication. The 28 year old son, later convicted for homicide, was admitted on the tenth day after the meal with very discrete neurological symptoms. This patient had low blood and urine thallium levels. He recovered uneventfully after treatment with Prussian Blue and forced diuresis. However, both surviving patients developed alopecia after two weeks. All three patients had raised liver transaminases. Intensive therapy with gut decontamination, Prussian Blue and forced diuresis should be the mainstay of therapy.

Tritium-labeled pyridoxamine, pyridoxal 5′-phosphate, and pyridoxine 5′-phosphate were prepared and administered intravenously to mice, and the distribution of isotope in liver and carcass between pyridoxine, pyridoxine 5′-phosphate, pyridoxal, pyridoxal 5′-phosphate, pyridoxamine and pyridoxamine 5′-phosphate was determined at different times after administration. The vitamin B6forms were extracted from the tissues by perchloric acid and separated by ion exchange chromatography. The results strongly suggest that phosphorylation is the first step in the conversion of pyridoxamine as well as of pyridoxine to the active coenzyme form of the vitamin. After an initial period of equilibration, pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate accounted for more than 90% of recovered isotope in liver and for 80 to 90% of recovered isotope in carcass. The ratio between these compounds was about 2:1 in both liver and carcass. About 10% of the total isotope in carcass appeared as pyridoxal. The available experimental data obtained after administration of different forms of vitamin B6were found to fit a kinetic metabolic model which assumes an equilibration between pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate but essentially unidirectional reactions in other metabolic conversions.

The sequential oxygenation of thymine to 5-hydroxymethyluracil, 5-formyluracil, and 5-carboxyuracil, coupled to the oxidative decarboxylation of 2-ketoglutarate, has been studied in 18O2experiments with an enzyme preparation from Neurospora crassastrain STA 4 (FGSC 262 A) and with ferrous ion, ascorbate, and catalase as cofactors. Three separate experiments were performed: (1) with thymine and 2-ketoglutarate, (2) with 5-hydroxymethyluracil and 2-ketoglutarate, and (3) with 5-formyluracil and 2-ketoglutarate. The 18O content was assessed by means of mass spectrometry after gas chromatographic separation of the trimethylsilyl derivatives of 5-hydroxymethyluracil (Experiment 1) and 5-carboxyuracil (Experiment 3) as well as of succinic acid (Experiments 1 to 3). The results show the incorporation of one atom of molecular oxygen in the products. It is concluded that the enzyme(s) catalyzing the three reactions belong(s) to the class of oxygenases for which a 2-keto acid functions for oxygen reduction.

Tritium-labeled pyridoxine was administered orally to one subject and intravenously to two subjects. The urinary excretion of isotope was followed daily for up to twenty-five days, and the specific radioactivity of the urinary 4-pyridoxic acid was determined. During the first days after administration of the pyridoxine, 15 to 20 per cent of the administered isotope was excreted in the urine; the remainder that was eliminated had a half-life of eighteen to thirty-eight days. A simple model for estimating the over-all metabolism of pyridoxine in human subjects is proposed, which consists of a small compartment with a rapid turnover rate and a larger storage compartment. The rate of elimination of vitamin B6from the body reservoir was estimated to be 2 to 3 per cent per day and the total turnover rate of the vitamin to be 2.2 to 4.4 per cent per day. Approximate values were calculated for the body content of the vitamin, 40 to 150 mg., and for the daily elimination, 1.7 to 3.6 mg. per day. The isotope-containing compounds in the urine were fractionated by ion exchange chromatography and it was found that 4-pyridoxic acid accounted for 20 to 40 per cent of the amount of isotope eliminated.

The half-life, pool size and turnover of cholic acid was determined in nine normal and four hypercholesteremic subjects after the administration of cholic acid-24-C14. The subjects were given a standardized frozen diet prepared with either butter or corn oil as the dietary fat. There was a considerable difference in the turnover of cholic acid between the different subjects but less variation when the same subject was studied twice on the same diet. The turnover cholic acid was higher in six of the normal subjects when they were given the corn oil-contaning version of the diet than when they were given the butter-containing version, but the differences were not statistically significant. In the hypercholesteremic subjects there was no significant effect of the diet on the turnover of cholic acid. It is concluded that the decrease in the concentration of serum cholesterol caused by the inclusion of mainly unsaturated fat in the diet was not caused by an increased formation of bile acids from cholesterol. In the normal subjects the half-life of cholic acid was 3.2 days on both types of diet, the pool size on the butter- and corn oil-containing versions was 1.12 and 1.30 gm., respectively, and the turnover was 0.296 and 0.314 gm per day, respectively.A paper chroinatographic technic was used to determine the ratio of glycine to taurine conjugated bile acids and the relative concentrations of cholic chenodeoxycholic and deoxycholic acids in duodenal bile. About 70 per cent of the bile acids were conjugated with glycine, irrespective of the type of diet. Cholic acid accounted for 34 to 39 per cent of the total bile acids, there being a tendency for lower values when the subjects were given the butter-containing version of the diet. Deoxycholic acid accounted for 25 to 29 per cent of the total bile acids in the normal subjects and for 14 to 16 per cent in the hypercholesteremic subjects.

The fecal excretion of total nitrogen and of total hexosamines has been determined in germfree and conventional rats. Germfree rats excreted more hexosamines than the conventional rats, while no difference in the nitrogen excretion was found. Infection of the germfree rats with a normal flora resulted in a temporarily increased excretion of hexosamines and nitrogen over a period of 2 to 3 days after which they reached the level of the conventional animals. The contents of the germfree cecum contained 65 to 137 mg of hexosamines and 57 to 127 mg of nitrogen as compared to 1.2 to 5.3 and 7.4 to 23 mg in conventional animals. The high figures for hexosamines were due to an increase in the total amount of contents in the cecum and to a fivefold increase in the concentration of hexosamine-containing material. Studies on the distribution of hexosamine-containing cecal contents between sediment and supernatant after centrifugation at 20,000 g for 2 hours demonstrated that 5 to 10 per cent of the hexosamines occurred in the sediment in the germfree rats, while 75 to 85 per cent was found in this fraction in the conventional rats. The soluble part of the cecal contents in germfree as well as in the conventional rats contained 70 per cent of hexosamines in molecules with a molecular weight above approximatively 100,000 as found by gel filtration experiments on sephadex gels. The higher weight of the germfree cecal wall was reflected in a high total amount of nitrogen and hexosamines. Isolated strains of bacteria capable of reducing the cecal size in vivo did not show any capacity to degrade the mucus in vitro in a test system, where a full intestinal flora was highly active.

IN TYROSINEMIA or tyrosinosis1 the main clinical features are multiple renal tubular defects as in the typical de ToniDebré Fanconi syndrome and nodular cirrhosis of the liver.1,2,3 Thrombocytopenia and increased pigmentation of the skin may be other characteristics. Slight mental retardation has been found in some of the patients.1 There are no particular neurologic abnormalities. Abdominal distension, rickets resistant to ordinary doses of vitamin D, or both have been the presenting symptoms at ages ranging between a few months and 4 years. The prognosis is variable. The disorder may cause death in early infancy, but some patients have also reached adult age. The causes of death have been liver failure or an acute water and electrolyte crisis due to the renal tubular defects.Biochemically, the condition which probably is inherited as an autosomal recessive gene is characterized by hypertyrosinemia and the urinary excretion of large amounts of

An enzyme preparation catalyzing the 2′-hydroxylation of thymidine was prepared from mycelial extracts of Neurospora crassastrain STA 4 (FGSC 262A) by chromatography on hydroxylapatite and DEAE-Sephadex A-50. The enzyme had an isoelectric point of 4.6 and a molecular weight of 47,000 (determined by gel filtration). The reaction product was identical with thymine riboside on ion exchange and thin layer chromatography. Paper chromatography after reduction and hydrolysis showed that the pentose moiety in the product was ribose. Of the pyrimidine deoxyribonucleosides tested as substrates, only those with oxygen at carbon atoms 2 and 4 were active, i.e.deoxyuridine, thymidine, 5-hydroxymethyldeoxyuridine, 5-bromodeoxyuridine, and 6-azathymidine. Thymidylate was attacked only slowly. Purine deoxyribonucleosides, deoxyribose, and 1-ethyluracil were not substrates. No evidence for control by nucleotides was obtained. The enzyme had an absolute requirement for ferrous ion and 2-ketoglutarate, and ascorbate and catalase were stimulatory. 2-Ketoglutarate was oxidatively decarboxylated during the reaction. Together with the previously demonstrated oxygen requirement (Shaffer, P. M., McCroskey, R. P., Palmatier, R. D., Midgett, R. J., andAbbott, M.T. (1968) Biochem. Biophys. Res. Commun.33, 806) the present findings establish that thymidine 2′-hydroxylase belongs to the group of oxygenases which require a 2-keto acid for oxygen reduction (Holme, E., Lindstedt, G., Lindstedt, S., andTofft, M. (1968) Fed. Eur. Biochem. Soc. Lett.2, 29; Lindblad, B., Lindstedt. G., Lindstedt, S., andTofft, M. (1969) J. Amer. Chem. Soc.91, 4604).