Infantile isolated sulphite oxidase deficiency: report of a case with negative sulphite test and normal sulphate excretion.

We present the clinical and biochemical data of a patient with infantile isolated sulphite oxidase deficiency with late onset of symptoms. A comparison of the biochemical parameters is made with the neonatal type of this disease and with the data of described patients with the combined defect of sulphite oxidase and xanthine oxidase, due to molybdenum cofactor deficiency. False-negative sulphite dip stick test as a pitfall in the diagnosis of sulphite oxidase deficiency is discussed.

Characterization of oxygen-resistant Chinese hamster ovary cells. III. Relative resistance of succinate and alpha-ketoglutarate dehydrogenases to hyperoxic inactivation.

We have recently shown that exposure of Chinese hamster ovary (CHO) cells to a toxic dose of normobaric hyperoxia (98% O2 for 3 days) caused a disturbance of cellular energy metabolism, that is, respiratory failure followed by stimulation of glycolytic activity and a net depletion of ATP. Respiratory failure was correlated with a selective inactivation of three mitochondrial enzymes, that is, partial inactivation of NADH dehydrogenase and virtually complete inactivation of succinate and alpha-ketoglutarate dehydrogenase activities (Schoonen et al., 1990). To elucidate the biochemical basis of resistance to hyperoxia in a previously described oxygen-resistant substrain of Chinese hamster ovary (CHO) cells, we compared the resistant cells with wildtype CHO cells with respect to several key parameters of oxidative and glycolytic energy metabolism. The two cell types were critically different in that the succinate and alpha-ketoglutarate dehydrogenases of the oxygen-resistant cells were relatively resistant to inactivation by hyperoxia, which may at least partly explain their enhanced capacity to respire and survive under hyperoxic conditions. Although the biochemical basis for the observed enzyme resistance to hyperoxic inactivation remains to be elucidated, the present data underscore the importance of succinate and alpha-ketoglutarate dehydrogenases as critical targets in hyperoxic killing of wildtype CHO cells.

Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia.

Continuous exposure of Chinese hamster ovary (CHO) cells to an atmosphere of 98% O2, 2% CO2 (normobaric hyperoxia) leads within a period of several days to cytostasis and clonogenic cell death. Here we report respiratory failure as an important early symptom of oxygen intoxication in CHO cells, resulting in a more than 80% inhibition of oxygen consumption within 3 days of hyperoxic exposure. This inhibition appeared to be correlated with selective inactivation of three mitochondrial key enzymes, NADH dehydrogenase, succinate dehydrogenase, and alpha-ketoglutarate dehydrogenase. The latter enzyme controls the influx of glutamate into the Krebs cycle and is particularly critical for oxidative ATP generation in most cultured cells, which depends on exogenous glutamine rather than glucose as a carbon source. As expected, the inactivation of alpha-ketoglutarate dehydrogenase was correlated with a fall in cellular glutamine utilization, which became apparent from the first day of hyperoxic exposure. Thereafter, glucose utilization and lactate excretion started to increase, up to 3-fold, indicating a cellular response to respiratory failure aimed at increased ATP generation from glycolysis. However, in spite of this response, the cellular ATP level progressively decreased, up to 2.5-fold. Thus, killing of CHO cells by normobaric hyperoxia seems to be due to a severe disturbance of mitochondrial metabolism eventually leading to a depletion of cellular ATP pools.

Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes.

Cellular intoxication by elevated concentrations of O2 may be considered as a model for accelerated cellular aging processes resulting from excessive free radical production by normal metabolic pathways. We describe here that exposure of HeLa cell cultures to 80% O2 for 2 days causes progressive growth inhibition and loss of reproductive capacity. This intoxication was correlated with inhibition of cellular O2 consumption and inactivation of 3 mitochondrial flavoproteins, i.e., partial inactivation of NADH and succinate dehydrogenases and total inactivation of alpha-ketoglutarate dehydrogenase. As alpha-ketoglutarate dehydrogenase controls the influx of glutamine/glutamate into the Krebs cycle, which is the major pathway for oxidative ATP generation in HeLa cells, the inactivation of alpha-ketoglutarate dehydrogenase was expectedly correlated with a net fall in glutamine/glutamate utilization. Furthermore, a simultaneous increase in glucose consumption and lactate production was observed, indicating that the cellular response to respiratory failure is to generate more ATP from glycolysis. In spite of this response, extensive depletion of ATP was observed. Thus, hyperoxia-induced growth inhibition and loss of clonogenicity seem to be due primarily to an impairment of mitochondrial energy metabolism resulting from inactivation of SH-group-containing flavoprotein enzymes localized at or near the inner mitochondrial membrane. These observations may be relevant for theories implicating loss of mitochondrial function as a prime factor in the aging process.

A new case of hyperargininaemia: neurological and biochemical findings prior to and during dietary treatment.

We present clinical and biochemical data on a further patient with hyperargininaemia and the results of neurophysiological tests both before and during dietary treatment with an essential amino acid mixture. With normalisation of plasma arginine concentrations, neurological functions improved and brain stem auditory evoked potentials normalized suggesting a partially reversible central conduction disorder. Neuroradiological findings included cerebral cortical atrophy on computed tomography scan and patchy abnormal myelination on magnetic resonance imaging (MRI). The typical clinical picture is discussed with reference to published therapeutical trials.

A milder variant of Zellweger syndrome.

A 4.5-year-old male patient is described with chorioretinopathy, minor facial anomalies, delayed closure of the fontanel, mental retardation, moderate hypotonia, epilepsy and hepatic fibrosis. Postural control, intentional vocalising and manual dexterity were superior to the performance of patients with classical Zellweger syndrome (ZS). Morphologically distinct peroxisomes were absent in the liver. In blood elevated pipecolic acid levels and abnormal levels of bile acid intermediates were found. The plasmalogen content of erythrocytes was normal. In fibroblasts we found an accumulation of very long chain fatty acids, decreased activity of acyl CoA:dihydroxyacetone phosphate acyltransferase, and impaired de novo biosynthesis of plasmalogens. On the basis of these clinical, ultrastructural and biochemical characteristics we assume that this patient represents a milder variant of the classical cerebro-hepato-renal syndrome of Zellweger.

Hydrogen breath test in schoolchildren.

The frequency of negative hydrogen breath tests due to colonic bacterial flora which are unable to produce hydrogen was determined after oral lactulose challenge in 98 healthy Dutch schoolchildren. There was a negative result in 9.2%. The probability of a false normal lactose breath test (1:77) was calculated from these results together with those from a separate group of children with lactose malabsorption (also determined by hydrogen breath test). A study of siblings and mothers of subjects with a negative breath test did not show familial clustering of this condition. Faecal incubation tests with various sugars showed an increase in breath hydrogen greater than 100 parts per million in those with a positive breath test while subjects with a negative breath test also had a negative faecal incubation test. The frequency of a false negative hydrogen breath test was higher than previously reported, but this does not affect the superiority of this method of testing over the conventional blood glucose determination.

New principle for estimation of hydrogen in expired air.

A new device for estimation of hydrogen in expired air is described. The measuring principle consists of a semi-conductive detector with a high affinity for hydrogen. Experiments on reliability and reproducibility are satisfactory. A good correlation has been obtained with the results of gas chromatographic analysis of hydrogen in reference gases as well as in samples of expired air. Discrimination between lactose absorbers and lactose malabsorbers in 50 consecutive patients is the same when using both methods simultaneously. This so-called "Lactoscreen", supplied with separate collection systems for infants and for older children, provides a rapid estimation of breath hydrogen concentration. A built-in hydrogen generator produces hydrogen air mixtures of variable concentrations, thus obviating the need for external reference gases. The "Lactoscreen" appears to be reliable for routine screening for carbohydrate malabsorption in children and adults and is easy for medical assistants to handle.

An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes.

An X-linked recessive disease is reported in a large pedigree. The disease is characterised by a triad of dilated cardiomyopathy, neutropenia and skeletal myopathy. The untreated patients, all boys, died in infancy or early childhood from septicemia or cardiac decompensation. Ultrastructural abnormalities were observed in mitochondria in cardiac muscle cells, neutrophil bone marrow cells and to a lesser extent (0-9%) in skeletal muscle cells. Membrane-bound vacuoles were seen in neutrophil bone marrow cells. Intramuscular fat droplets were increased in type I skeletal muscle fibres. An affected patient had intermittent lactic acidemia, borderline low plasma carnitine, the latter decreasing during periods of illness, and low muscle carnitine (27% pretreatment; 35-40% posttreatment). While on treatment with oral carnitine he had less weakness and no cardiac complaints, but his neutropenia was not affected. Respiratory chain abnormalities were observed in this patient's isolated skeletal muscle mitochondria. These were: (1) diminished concentrations of cytochromes c1 + c, b and aa3 to 29, 47 and 64% of the averaged controls, and (2) a lowered P:0 ratio for oxidation of ascorbate + TMPD, with diminished uncoupler stimulated Mg2+-ATPase activity. Muscle AMP deaminase was deficient (5 resp. 17%). Only one previous report (Neustein et al. 1979) on X-linked mitochondrial cardiomyopathy exists, which probably refers to the same entity. Biochemical studies and haematological abnormalities (neutropenia) are reported for the first time.

Physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues in relation to glycogenosis type II (Pompe's disease).

The physico-chemical and immunological properties of acid alpha-glucosidase from various human tissues have been studied. Heat stability of acid alpha-glucosidase from heart, liver and skeletal muscle is identical, but for kidney some different results are obtained. Identical isoelectrofocussing patterns are found for heart, liver and skeletal muscle. Furthermore, the effect of antiserum against human liver acid alpha-glucosidase on the activity of acid alpha-glucosidase from various tissues is studied. The results are discussed in relation to glycogenosis type II (Pompe's disease).