Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger.

Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was only slow incorporation of radioactivity into 24-OH-THCA and into the C29-dicarboxylic acid. From the specific activity decay curve of 14C in cholic acid obtained after intravenous injection of 14C-cholic acid, the pool size of cholic acid was calculated to be 24 mg/m2 and the daily production rate to 9 mg/m2 per d. This corresponds to a reduction of approximately 85 and 90%, respectively, when compared with normal infants. It is concluded that liver peroxisomes are essential in the normal conversion of THCA to cholic acid. In the Zellweger syndrome this conversion is defective and as a consequence the accumulated THCA is either excreted as such or transformed into other metabolites by hydroxylation or side chain elongation. The accumulation of THCA, as well as the similar rate of conversion of 5 beta-cholestane-3 alpha,7 alpha.12 alpha-triol and THCA into cholic acid, support the contention that the 26-hydroxylase pathway with intermediate formation of THCA is the most important pathway for formation of cholic acid in man.

Urinary excretion of dicarboxylic acids from patients with the Zellweger syndrome. Importance of peroxisomes in beta-oxidation of dicarboxylic acids.

The urinary excretion of adipic acid, suberic acid and sebacic acid from two patients with the cerebrohepato-renal syndrome of Zellweger was studied. The patients had a complete lack of peroxisomes in the liver as judged by electron microscopy. In the non-ketotic state, the total excretion of free and conjugated adipic acid, suberic acid and sebacic acid was increased by about 100%, 200% and 350%, respectively, as compared to the corresponding excretion from six healthy infants of the same age. The excretion of free dicarboxylic acid was increased to a considerably lesser extent than the free + conjugated dicarboxylic acid. In view of the presence of adipic acid in urine of the Zellweger patients, it is concluded that peroxisomes are not obligatory for beta-oxidation of medium-chain dicarboxylic acids in vivo. The relative accumulation of suberic acid and sebacic acid as compared to adipic acid is, however, consistent with a relative block in the conversion of suberic acid and sebacic acid into adipic acid in patients with the Zellweger syndrome.

Plasma tocopherol levels and vitamin E/beta-lipoprotein relationships during pregnancy and in cord blood.

Plasma tocopherol, beta-lipoprotein concentrations, and tocopherol/beta-lipoprotein ratios were studied during 40 normal pregnancies. The levels in 36 cord blood samples from the newborn of these pregnancies and in 25 normal nonpregnant women were also determined. In agreement with earlier studies plasma tocopherol levels rose gradually and significantly during pregnancy, while the levels in cord blood were much lower. Beta-lipoprotein concentrations showed similar changes as for tocopherol, rendering the tocopherol/beta-lipoprotein ration unchanged during gestation. The ratios in cord blood and in nonpregnant women were similar to those of pregnant women. A significant positive correlation (r=0.84, p less than 0.001) was found between tocopherol and beta-lipoprotein concentrations. The results indicate that the increased plasma tocopherol levels during pregnancy and the low levels in cord blood result from differences in plasma transport capacity.

Plasma ferritin concentrations in preterm infants in cord blood and during the early anaemia of prematurity.

Ferritin concentrations in cord blood were determined in 22 normal term and 32 preterm infants (birth weights 600-2000 g). Eight of the preterms were SGA infants. AGA preterm infants had significantly lower concentrations than term infants, and the SGA preterm newborn had even lower levels. Plasma ferritin in cord blood of the term and AGA preterm infants correlated positively with plasma iron and transferrin saturations, but not with the transferrin level, while plasma iron and transferrin concentrations correlated positively. In a longitudinal study, 17 AGA preterm infants (birth weights 850-1500 g) were followed during the early anaemia of prematurity. Iron was supplemented from 4 weeks of age. Plasma ferritin rose rapidly during the first days after birth, peak levels being reached at 1-4 weeks. Thereafter linear falls (semilog) occurred with similar slopes in different infants. Transferrin concentrations showed a slow progressive increase from 0-8 weeks. Plasma ferritin, after reaching the peak value, correlated negatively with weight gain. No infant had low ferritin values indicating iron deficiency during the early anaemia.

Ceruloplasmin levels and erythrocyte superoxide dismutase activity in small preterm infants during the early anemia of prematurity.

Ceruloplasmin plasma levels and erythrocyte superoxide dismutase activity were studied in appropriate for gestational age preterm infants (birth weights less than or equal to 1500 g) during the first 10 weeks of life. Preterm infants had significantly lower ceruloplasmin concentrations in cord blood than term infants, the mean level in the preterm infants being 0.07 g/l. At 1 week of age ceruloplasmin levels had risen significantly, whereupon a fall occurred at 2 weeks of age. Ceruloplasmin concentrations increased slowly and progressively from 4 weeks of age. The low ceruloplasmin concentration during the early anemia of prematurity seems not to interfere with iron mobilization. The superoxide dismutase activity per gram hemoglobin in cord blood erythrocytes from normal term infants was significantly lower than that of red blood cells from adults. When the activity was expressed per erythrocyte no difference was found. The normochromic macrocytic red blood cells of the neonate most likely explain this discrepancy. The erythrocyte superoxide dismutase activity of the preterm infants did not change from birth until 10 weeks of age, and the levels seemed adequate judged from the levels found in red blood cells from adults and cord blood from term infants. Neither ceruloplasmin nor erythrocyte superoxide dismutase activity seem to play a role in the etiology of the early anemia of prematurity.

[Normal hemoglobin levels in children with cyanotic heart disease. Is it iron deficiency anemia?]. / "Normal" hemoglobinverdi hos barn med cyanotisk hjertefeil. Er det jernmangelanemi?

Decreased arterial oxygen saturation in cyanotic congenital heart disease causes a compensatory rise in haemoglobin and haematocrit levels. There is an inverse correlation between arterial oxygen saturation and haemoglobin/haematocrit. This holds true as long as the erythropoiesis is not restricted by other factors. The haematological values and arterial oxygen saturations of three children with cyanotic congenital heart disease are presented. They illustrate how iron deficiency causes discrepant values for arterial oxygen saturation and haemoglobin/haematocrit and that "normal" haemoglobin/haematocrit levels in such children may constitute anaemia. Measurements of MCV, MCH and serum ferritin reveal the existence of iron deficiency anaemia. Low grade iron medication is recommended for children with cyanotic congenital heart disease.

In vitro assay for erythropoietin: erythroid colony formation in methyl cellulose used for the measurement of erythropoietin in plasma.

Erythroid colony formation in methyl cellulose has been used for the measurement of erythropoietin in plasma. Livers from newborn mice less than 24 hr old were found to provide convenient target cells. Newborn mouse liver contains a substantial number of erythroid colony-forming cells (CFU-e) that have a high sensitivity to erythropoietin, the dose--response curve for erythropoietin reaching a plateau at 50 mU/ml. As little as 0.5 m/ml of the hormone is detectable. Removal of cells that adhered to glass prior to culturing doubled the number of colonies formed in the presence of erythropoietin. Addition of untreated plasmas that showed high erythropoietin titers in the exhypoxic polycythemic mice assay gave variable results. Some of the plasmas stimulated colony formation actively and in a linear fashion. However, the majority of the plasmas were toxic to the cultures. Dialyzing the plasmas for 3 days against distilled water effectively removed the toxicity. Results obtained with the method are in good agreement with the values found using the exhypoxic polycythemic mice assay.

Selenium and vitamin E in cord blood from preterm and full term infants.

Selenium was determined in erythrocytes and serum, and vitamin E and beta-lipoprotein in serum from cord blood samples of 31 full term and 20 preterm infants. Venous samples from 21 mothers at birth and 15 normal adult women were also analyzed. No difference for either selenium or vitamin E was found between the preterm and full term infants. The selenium concentration in red blood cells was the same for newborn, mothers at birth, and normal adult women. The serum concentration of selenium was, however, significantly lower in the newborn, the mean level in the children being 64% of that in the mothers. The level in the mothers did not differ from that in non-pregnant women. The vitamin E concentration was found to correlate very well with the beta-lipoprotein concentration. This indicates that differences in the transport capacity account for the large difference in the serum tocopherol levels of mothers at birth and newborn.

Plasma erythropoietin levels in mice. Response to hypoxia at different ages.

Mice from newborn until adult age were exposed to hypobaric hypoxia of 0.5 atm for 6 h. In all age groups the animals responded with increased plasma erythropoietin (Ep) levels. A greater response was elicited from adult mice compared to newborn animals. No sex difference in response occurred. Twelve hours after the hypoxic exposure no plasma Ep activity was detectable. Though high plasma Ep levels were present in non-hypoxic mice 9 and 20 days old, hypoxia further increased the Ep activity. It is concluded that hypoxia stimulates Ep production during hepatic as well as during erythropoiesis. Though erythroid target cells may be maximally stimulated by Ep in the young mouse (9-20 days old) a reverse capacity for Ep production exists. Increased capacity for Ep production may develop towards adult age.

[Water intoxication with hyponatremia and cramps in the newborn period]. / Vannintoksikasjon med hyponatremi og kramper i nyfødtperioden.

Acute water intoxication with hyponatraemic convulsions has been described several times in children less than two years old. We describe a two-day-old boy who developed hyponatraemia with convulsions because of water intoxication during his stay in the maternity ward. It was a hot day, and the baby was observed to sweat abundantly. The mother therefore breast fed him at frequent intervals and in addition offered him plain water, which he drank eagerly, approximately 350 ml in the course of a few hours. He then developed convulsions with bilateral clonic flexion movements of the arms, and serum sodium concentration was 113 mmol/l (114 on repeat). He was treated with diazepam and phenobarbitone and 20 mmol NaCl as slow infusion, and serum Na was 135 mmol/l after 20 hours. During treatment he passed large amounts of dilute urine and lost 110 g of weight. No abnormal loss of salt was found, and follow-up for six months has been uneventful. Neonates are at risk of developing hyponatraemia and convulsions when offered large amounts of electrolyte poor solutions, especially during hot weather.

[Primary pulmonary hypertension in children]. / Primaer pulmonal hypertensjon hos barn.

Primary pulmonary hypertension is a clinical syndrome with severe pulmonary hypertension where other causal diseases are excluded. The condition is progressive, fatal and disappointingly resistant to therapy. In this article we present three children with the disease. Two of the patients died shortly after diagnosis, one of them during heart catheterization. The third patient showed good response to treatment with calcium channel blockers, diuretics and nightly supplementary oxygen. Quality of life and hemodynamics improved during the first six months of treatment, and remained virtually unchanged for another two years. Her condition then deteriorated rapidly, and she died three years after diagnosis. According to the literature and our experience, investigations with non-invasive methods such as oxygen saturation measurement, echocardiography, and a simple exercise test provide sufficient information to start therapy and monitor therapeutic response. Some patients show vasodilatory response to treatment with drugs, and might benefit from therapy with calcium channel blockers.

Plasma erythropoietin levels in mice during the growth period.

Plasma erythropoietin (erythropoiesis stimulating factor(s), ESF), PCV and body weight were measured in normal mice from birth until the age of 70 d. Low but detectable ESF activity was present at birth, following by undetectable levels 12-48 h after birth. Thereafter the ESF level rapidly increased. Peak levels were obtained 15-20 d after birth. During the same phase an increased growth velocity occurred, coincident with decreased PCV levels. A fall to undetectable levels in plasma ESF activity coincided with decelleration of growth 40-50 d after birth. In small litters (four pups per litter) growth velocity and PCV levels were significantly higher than in large litters (16 pups per litter) 8 and 15 d after birth. The plasma ESF activity, however, did not differ between the two groups. Prohibiting suckling for a period of 16 h did not change the plasma ESF level. The hypothesis is put forward that growth directly or indirectly stimulates ESF production.

The regulation of erythropoiesis in protein-energy-malnutrition.

The erythropoietin (ESF) content of plasma and urine has been studied in children with protein-energy-malnutrition (PEM) living in the Kivu province at an altitude of 1467--2000 m. On admission, packed cell volume (PCV) was moderately reduced; after 2 months of refeeding PCV had increased but was still lower than in the controls. Plasma ESF was increased on admission and in patients refed for 2 months. The expected positive correlation between serum and urine ESF levels was found after refeeding but not on admission; the last finding could not be explained by the presence of erythropoiesis inhibiting factor(s) in the urine. In spite of the normal 2,3-DPG and P50 previously described in PEM in Kivu, the anaemia associated with this disease is not merely an adaptation to lowered oxygen consumption and there must be some disturbances in the responsiveness of bone marrow to ESF. The high ESF values observed after 2 months of refeeding confirm that the restoration of the red cell volume proceeds slowly.

Plasma erythropoietin concentrations during the early anemia of prematurity.

Plasma erythropoietin concentrations were studied in 11 preterm appropriate for gestational age infants at the age of 3-14 weeks. Their birth weights ranged from 860-1 690 g. Erythropoietin was measured by a cell culture technique. Significant concentrations of erythropoietin was detected in 18 out of 29 samples, at all stages of the early anemia. The highest levels were found at 3-9 weeks. Individual erythropoietin values did not correlate with hemoglobin concentrations, hematocrit levels or 'corrected' reticulocyte counts, nor did the 'corrected' reticulocyte count correlate with hemoglobin or hematocrit. The lack of correlation with hemoglobin concentration most likely reflects the importance of other factors as well as the hemoglobin in determining the oxygenation status of the infant. A significant positive correlation (r = 0.60, p less than 0.01) was found between erythropoietin concentration and weight gain in the preceding week. The study shows that small preterm infants are capable of erythropoietin production during their early anemia, and indicates that the hormone plays a role in the regulation of erythropoiesis also at this time of life.

Serum immunoreactive erythropoietin in healthy normal children.

To provide reference data for normal children, serum immunoreactive erythropoietin (siEp) was estimated by radioimmunoassay in samples from 130 healthy children, 57 girls and 73 boys, with ages between 1 month and 16 years. In 128 of the children the (geometric) mean siEp was 15.8 miu/ml (iu: international units) with 95% range (the range within which 95% of the observations are predicted to fall) 9.1-27.6 miu/ml. There was no relation between siEp and the variables haemoglobin (Hb), PCV, age and sex. There were two outliers, both girls, aged 9.5 and 9.8 years, in whom siEp was greater than 256 miu/ml. In both, Hb and PCV were normal and we are unable to account for these atypical findings. Estimates of siEp in the 128 children were not significantly different from those in 22 healthy adults investigated simultaneously (mean 16.2 miu/ml, with 95% range 11.2-23.3 miu/ml).

Congestive heart failure caused by vitamin D deficiency?

We describe a child, 3.5 months old, with severe vitamin D deficiency, profound hypocalcaemia, hyperphosphataemia, dilated left ventricle, severely reduced myocardial contractility and congestive heart failure. She also had depressed thyroid function with subnormal thyroxine and non-detectable serum thyrotropin (TSH) levels. The child promptly responded to calcium infusions, conventional anticongestive therapy and calcitriol. She is now 3 years old and received no medication. Myocardial function is normal but she has motor delay. We believe that her transitory congestive heart failure was caused by severe vitamin D deficiency with profound hypocalcaemia.