Peritoneal protein losses and cytokine generation in automated peritoneal dialysis with combined amino acids and glucose solutions.

OBJECTIVES: Protein-energy malnutrition as a consequence of deficient protein intake frequently occurs in peritoneal dialysis (PD) patients. Previously, we showed that peritoneal dialysate containing a mixture of amino acids (AA) and glucose has anabolic effects. However AA-dialysate has been reported to increase intraperitoneal protein and AA losses and the release of proinflammatory cytokines (interleukine-6 (IL-6) and tumor necrosis factor alpha (TNFalpha)). We investigated the effect of AA plus glucose (AAG) solutions on peritoneal protein losses and cytokine generation. METHODS: In 6 patients on standard automated peritoneal dialysis (APD) 12 APD sessions of 6 cycles each were performed during the night using dialysate containing 1.1% AA plus glucose or glucose alone as control. Protein losses and TNFalpha and IL-6 concentrations were measured in dialysates separately collected from nightly cycling and daytime dwell. RESULTS: The 24 hour-protein losses with AAG (median 6.7 g, range 4.7-9.4 g) were similar to control dialysate (median 6.0 g, range 4.2-9.2 g). Daytime dialysate IL-6 levels were higher after nightly AAG dialysis than after control dialysis (142 pg/ml and 82 pg/ml, respectively, P<.05). TNFalpha concentrations were very low. CONCLUSION: Nightly APD with amino acids containing dialysate was associated with an increase in peritoneal IL-6 generation during the day. The addition of AA to standard glucose dialysis solutions did not induce a significant increase of peritoneal protein losses.

[Lactic acidosis and accumulation of glutamate in the blood of neonates following treatment with calcium levulinate for hypocalcaemia]. / Lactaatacidose en glutamaatophoping in het bloed van neonaten na behandeling met calciumlevulaat wegens hypocalciëmie.

The data from 5 clinics concerning 8 infants, who had developed severe lactic acidosis and hyperglutamic acidaemia were reviewed. Blood-lactate levels were up to 15 mmol/l (reference level: < 2) and plasma-glutamate levels up to 1632 pmol/l (reference level: 14-78), and there was no concomitant hyperglutaminaemia (levels up to 1032 micromol/l (reference level: 333-809)). A positive correlation between the amount of calcium levulinate administered and the degree of hyperglutamic acidaemia was found. Replacement of the calcium levulinate by another calcium salt caused a reversal of the biochemical abnormalities of the patients. Two of the infants had a 22q11 microdeletion. This development of severe acidosis in infants who had been given a calcium supplement in the form of calcium levulinate may be related to genetic predisposition. The paradoxal hyperketonaemia and generalized aminoaciduria in 4 other patients suggested disturbed function ofthe mitochondrial respiratory chain. The hypothesis of the occurrence of an underlying defect of the mitochondrial respiratory chain was tested in the muscle tissue of one 22q11 patient, but this showed no abnormalities. Excessive accumulation of glutamate because of dysfunction ofglutamine synthetase, which forms glutamate from glutamine seems unlikely because of the relatively low values of plasma glutamate compared to the glutamine plasma levels. Calcium levulinate should no longer be used in neonates as it may lead to lactic acidosis.

[Diagnosis of vitamin B12 deficiency revised]. / De diagnostiek van vitamine-B12-deficiëntie herzien.

Vitamin B12 (cobalamin) deficiency is a common disorder with potential irreversible haematological and neurological consequences. Currently used diagnostic tests such as the evaluation of serum vitamin B12 and the Schilling test are insufficient, e.g. the positive predictive value of a low serum vitamin B12 level for actual vitamin B12 deficiency (i.e. tissue deficiency) is low. Insufficient availability of vitamin B12 will lead to the accumulation of methylmalonic acid and homocysteine in the body. Nearly all patients with vitamin B12 deficiency also have substantially increased levels of methylmalonic acid and homocysteine. New tests of serum methylmalonic acid and homocysteine are highly sensitive for vitamin B12 deficiency and may obviate the need for the somewhat cumbersome Schilling test.

Genomic organization of the human phosphomannose isomerase (MPI) gene and mutation analysis in patients with congenital disorders of glycosylation type Ib (CDG-Ib).

CDG-Ib is the "gastro-intestinal" type of the congenital disorders of glycosylation (CDG) and a potentially treatable disorder. It has been described in patients presenting with congenital hepatic fibrosis and protein losing enteropathy. The symptoms result from hypoglycosylation of serum- and other glycoproteins. CDG-Ib is caused by a deficiency of mannose-6-phosphate isomerase (synonym: phosphomannose isomerase, EC 5.3.1.8), due to mutations in the MPI gene. We determined the genomic structure of the MPI gene in order to simplify mutation detection. The gene is composed of 8 exons and spans only 5 kb. Eight (7 novel) different mutations were found in seven patients with a confirmed phosphomannose isomerase deficiency, analyzed in the context of this study: six missense mutations, a splice mutation and one insertion. In the last, the mutation resulted in an unstable transcript, and was hardly detectable at the mRNA level. This emphasizes the importance of mutation analysis at the genomic DNA level.

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.

Different effects of cycloheximide and chloramphenicol on corticosterone production by isolated rat adrenal cells.

Cycloheximide and chloramphenicol both inhibit the stimulating effect of adenocorticotropic hormone (ACTH) on adrenal steroid production. To test whether these inhibitors had andy effect on adrenal steroid production, independent fromthe mechanism of action of ACTH we investigated their effect on the conversion of 25-hydroxycholesterol into corticosterone in isolated rat adrenal cells. Cycloheximide, both in the absence and in the presence of ACTH, had no effect on this conversion. Chloramphenicol inhibited the conversion of 25-hydroxycholesterol into corticosterone whether ACTH has no direct efeect on the cholesterol side-chain cleaving system. The inhibition by chloramphenicol of the ACTH-stimulated steroid production is at least partly due to inhibition of one or more of the processes involved in the conversion of 25-hydroxycholesterol into corticosterone.

beta-Glucuronidase deficiency as a cause of fetal hydrops.

Mucopolysaccharidosis type VII (MPS VII) was diagnosed in a case of severe fetal hydrops. beta-glucuronidase deficiency was demonstrated in the amniotic fluid, which was obtained at 25 weeks of gestation, and in the fibroblasts of the child, which were cultured after fetal death in the 26th week of gestation. In the amniotic fluid the two-dimensional electrophoresis pattern of glycosaminoglycans was in agreement with MPS VII. Based on these results, prenatal diagnosis could be offered to the couple for the next pregnancy.

Prenatal diagnosis of the urea cycle diseases: a survey of the European cases.

A European survey of prenatal diagnosis cases involving urea cycle diseases was performed. Citrullinemia was the most frequently investigated disease (108 cases). Other diseases are, in order of frequency, argininosuccinic aciduria (75 cases), ornithine transcarbamylase defect (52 cases), carbamoylphosphate synthetase defect (8 cases), triple H (3 cases), and arginase deficiency (1 case). Only one disease (ornithine transcarbamylase defect) is presently diagnosed using molecular biology methods.

Morquio B syndrome: a primary defect in beta-galactosidase.

Fibroblasts from patients with Morquio B syndrome contain normal numbers of beta-galactosidase molecules with normal turnover but strongly reduced activity per enzyme molecule. Various substrate affinities are abnormal: the Km for methylum belliferyl (MU)-beta-galactoside is 4-10-fold elevated and affinity for keratan sulphate and oligosaccharides, isolated from Morquio B urine, was not detectable. In contrast, these substrate affinities are normal for beta-galactosidase in adult type GM1-gangliosidosis fibroblasts. Cell hybridization studies demonstrate that Morquio B syndrome and infantile and adult type GM1-gangliosidosis belong to the same complementation group. From these results we conclude that Morquio B syndrome is caused by a mutation in the structural gene for beta-galactosidase, which is allelic to the mutations in infantile and adult type GM1-gangliosidosis. Urinary excretion of keratan sulphate and oligosaccharides is abnormal in Morquio B syndrome but normal in adult type GM1-gangliosidosis. The catalytic properties of beta-galactosidase in Morquio B syndrome and GM1-gangliosidosis provide a possible explanation for the distinct clinical manifestations in these disorders.

Identification of glycosaminoglycans in age-related macular deposits.

We investigated the presence and localization of glycosaminoglycans in basal laminar deposit and drusen in age-related maculopathy. Conventional histological staining techniques and monoclonal antibodies specific for several glycosaminoglycans were used on paraffin-embedded human maculae. Furthermore, macular homogenates were analyzed with two-dimensional electrophoresis. Quantitative analysis of glycosaminoglycans was done spectrophotometrically using dimethylmethylene blue. Immunohistochemically, all basal laminar deposit stained positive for chondroitin 4-sulfate and focally positive for heparan sulfate proteoglycan. Drusen were not stained with any of the monoclonal antibodies. With two-dimensional electrophoresis, it was demonstrated that macular extracts with and without age-related maculopathy contained chondroitin sulfate. Heparan sulfate was only expressed in maculae with age-related maculopathy. The total amount of glycosaminoglycans was significantly higher in maculae with basal laminar deposit than in maculae without basal laminar deposit (P = .001). There were significant differences in the amount and composition of glycosaminoglycans between maculae with and without age-related maculopathy.

Amino acid solutions for premature neonates during the first week of life: the role of N-acetyl-L-cysteine and N-acetyl-L-tyrosine.

Tyrosine and cyst(e)ine are amino acids that are thought to be essential for preterm neonates. These amino acids have low stability (cyst(e)ine) or low solubility (tyrosine) and are therefore usually present only in small amounts in amino acid solutions. Acetylation improves the stability and solubility of amino acids, facilitating a higher concentration in the solution. We compared three commercially available amino acid solutions, Aminovenös-N-päd 10%, Vaminolact 6.5%, and Primène 10%, administered to 20 low-birth-weight neonates on total parenteral nutrition from postnatal day 2 onward. Aminovenös-N-päd 10% contains acetylated tyrosine and acetylated cysteine; the other solutions do not contain acetylated amino acids and differ in the amount of tyrosine and cysteine added. On postnatal day 7, plasma amino acids were measured together with urinary excretion of amino acids and the total nitrogen excretion; 38% of the intake of N-acetyl-L-tyrosine and 53% of the intake of N-acetyl-L-cysteine were excreted in urine. Plasma levels of N-acetyl-L-tyrosine (331 +/- 74 mumol/L) and N-acetyl-L-cysteine (18 +/- 29 mumol/L) were higher than those of tyrosine (105 +/- 108 mumol/L) and cystine (11 +/- 9 mumol/L), respectively. Plasma tyrosine levels in the groups receiving small amounts of tyrosine remained just below the reference range. We show a linear correlation of plasma cystine with the intake of cysteine (r = .75, p = 0.01), but not with N-acetyl-L-cysteine. The estimated intake of cysteine should be 500 mumol.kg-1.d-1 in order to obtain levels comparable with those shown in normal term, breast-fed neonates. Nitrogen retention did not differ among the three groups (247 to 273 mg.kg-1.d-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Albumin and whole-body protein synthesis respond differently to intraperitoneal and oral amino acids.

Patients with peritoneal dialysis are at risk for malnutrition and hypoalbuminemia, which are indicators of poor outcome. Recently, it was shown that dialysis solutions containing amino acids (AAs) and glucose improve protein anabolism in peritoneal dialysis patients. We determined if the same solutions could increase the fractional synthesis rate of albumin along with whole-body protein synthesis. Changes in the fractional albumin synthetic rate reflect acute change in hepatic albumin synthesis. A random-order cross-over study compared the effects of Nutrineal (AA source) plus Physioneal (glucose) dialysate with Physioneal alone dialysate. Eight patients in the overnight fasting state were compared to 12 patients in the daytime-fed state. Fractional albumin synthetic rate and whole-body protein synthesis were determined simultaneously using a primed-continuous infusion of L-[1-(13)C]-leucine. Fractional albumin synthesis on AAs plus glucose dialysis did not differ significantly from that on glucose alone in the fasting or the fed state. Protein intake by itself (fed versus fasting) failed to induce a significant increase in the fractional synthetic rate of albumin. Conversely, the oral protein brought about a significant stimulation of whole-body protein synthesis. Our findings show that the supply of AAs has different effects on whole-body protein synthesis and the fractional synthetic rate of albumin.

Brain abnormalities in a case of malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase (MCD) deficiency is an extremely rare inborn error of metabolism that presents with metabolic acidosis, hypoglycemia, and/or cardiomyopathy. Patients also show neurological signs and symptoms that have been infrequently reported. We describe a girl with MCD deficiency, whose brain MRI shows white matter abnormalities and additionally diffuse pachygyria and periventricular heterotopia, consistent with a malformation of cortical development. MLYCD-gene sequence analysis shows normal genomic sequence but no messenger product, suggesting an abnormality of transcription regulation. Our patient has strikingly low appetite, which is interesting in the light of the proposed role of malonyl-CoA in the regulation of feeding control, but this remains to be confirmed in other patients. Considering the incomplete understanding of the role of metabolic pathways in brain development, patients with MCD deficiency should be evaluated with brain MRI and unexplained malformations of cortical development should be reason for metabolic screening.

Two novel mutations in SLC6A8 cause creatine transporter defect and distinctive X-linked mental retardation in two unrelated Dutch families.

Four Dutch male patients, two brothers from unrelated families were referred for investigation of psychomotor and severe language/speech delay. All four patients showed growth deficiency over the years. Facial features and poor body habitus were quite similar in the patients and in their mothers. Brain MRI showed nonspecific periventricular white matter lesions. In all the patients neuropsychological tests revealed moderate mental retardation, attention deficit and hyperactivity with impulsivity, a semantic-pragmatic language disorder, and oral dyspraxia. This specific cognitive profile is different from other children with mental retardation syndromes and seems to be unique. Excretion of creatine to creatinine ratio in urine of the four boys was increased compared to controls and their creatine uptake in fibroblasts was deficient. In the two brothers from the first pedigree, DNA sequence analysis revealed a novel mutation in the splice donor site in intron 10 (IVS10 + 5G>C, c.1495 + 5G>C) of the SLC6A8 gene leading to skipping of exon 10. In the other sib pair a novel missense mutation (c. 1361C>T; p.Pro544Leu) was found. These are the first families reported, in which the clinical suspicion of a creatine transporter disorder was raised on clinical grounds, before a brain 1H-MRS suggested the diagnosis. Screening of apparently X-linked mental retarded patients with this somatic and behavioral phenotype by the biochemical assay of creatine to creatinine ratio in the urine or DNA sequence analysis of SLC6A8 is worthwhile even when 1H-MRS is not available.

Differential diagnosis of (inherited) amino acid metabolism or transport disorders.

Disorders of amino acid metabolism or transport are most clearly expressed in urine. Nevertheless the interpretation of abnormalities in urinary amino acid excretion remains difficult. An increase or decrease of almost every amino acid in urine can be due to various etiology. To differentiate between primary and secondary aminoacido-pathies systematic laboratory investigation is necessary. Early diagnosis of disorders of amino acid metabolism or transport is very important, because most of them can be treated, leading to the prevention of (further) clinical abnormalities. In those disorders, which cannot be treated, early diagnosis in an index-patient may prevent the birth of other siblings by means of genetic counseling and prenatal diagnosis.Primary aminoacidopathies can be due to genetically determined transport disorders and enzyme deficiencies in amino acid metabolism or degradation. Secondary aminoacidopathies are the result of abnormal or deficient nutrition, intestinal dysfunction, organ pathology or other metabolic diseases like organic acidurias.A survey of amino acid metabolism and transport abnormalities will be given, illustrated with metabolic pathways and characteristic abnormal amino acid chromatograms.

Effect of 25-hydroxy-cholesterol on the uptake of corticosterone in isolated rat liver cells.

Isolated rat liver cells are able to take up corticosterone. The Arrhenius plot of the uptake shows a biphasic course with a change in the slope around 25 degrees C. 25-OH-cholesterol is also taken up; this phenomenon reaches a maximum at 10-15 minutes. After preincubating liver cells at 37 degrees C in the presence of this sterol the phase transition is shifted to a higher temperature (32 degrees C) as shown in the Arrhenius plot of the corticosterone uptake. At the same time the uptake of corticosterone is diminished. This cannot readily be explained by direct competition. The mechanism might involve an inhibition of an active uptake mechanism caused by a change in the plasma membrane.

Effects of some hydroxylated sterols on the steroid production in isolated rat adrenal glomerulosa and fasciculata/reticularis cells in the presence of potassium, angiotensin II or ACTH.

22S-OH-cholesterol and 25-OH-cholesterol are good aldosterone precursors in isolated rat adrenal glomerulosa cells. In the two concentrations tested (25 and 50 microM) 25-OH-cholesterol stimulated the aldosterone production in a (not-linear) dose-dependent way. An increase in the 22S-OH-cholesterol concentration from 25 microM to 50 microM led to a decrease in the aldosterone production. The exogenous substrate deoxycorticosterone, entering the steroidogenic pathway after the cholesterol side-chain cleavage, is a much better substrate than the sterols mentioned. These results suggest that the cholesterol side-chain cleavage is the rate-limiting step in the aldosterone production from both sterols. We found no effect of the sterols on the potassium-induced aldosterone synthesis. This might be explained by the existence of separate pools of steroid intermediates within the adrenal cell. In vitro a difference in steroid production rates exists between glomerulosa and fasciculata/reticularis cells. This may arise from differences in availability of endogenous steroid precursors like cholesterol. However similar differences can be observed if exogenous substrates like 25-OH-cholesterol or 22S-OH-cholesterol are used. These results therefore suggest that enzymatic activities in the steroidogenic pathway are more important than the cholesterol concentration in regulating steroid production in isolated rat adrenal glomerulosa and fasciculata/reticularis cells.

Conversion of 22S-hydroxy-cholesterol and its effects on the metabolism of other sterols in rat adrenal cells and bovine adrenal mitochondria.

This study provides evidence that 22S-OH-cholesterol inhibits the conversion of 25-OH-cholesterol but has no effect on the conversion of 22R-OH-cholesterol. The latter sterol is an intermediate in the cholesterol side-chain cleavage, whereas for the conversion of 25-OH-cholesterol into pregnenolone the complete side-chain cleaving enzyme system is necessary. This complements a previous study in which it was shown, that 22S-OH-cholesterol has an inhibitory effect on the ACTH-induced conversion of cholesterol into corticosterone in isolated rat adrenal cells. The available evidence thus suggests an inhibition by 22S-OH-cholesterol of the first step in the cholesterol side-chain cleavage. The results, obtained from the experiments with rat adrenal cells and with bovine adrenal mitochondria, allow the hypothesis, that a causal relationship exists between conversion of 22S-OH-cholesterol and production of corticosterone, respectively pregnenolone. We conclude, that 22S-OH-cholesterol is a substrate for steroid production in the adrenal cell. This sterol inhibits the ACTH-stimulated corticosterone production. The site of this inhibition is located at one of the first steps in the cholesterol side-chain cleavage, probably the binding of cholesterol to the cytochrome P450-complex.

A clinical biochemist's view of the investigation of suspected inherited metabolic disease.

The necessity for a multi-disciplinary approach to the study of genetic disease is discussed. The progress of laboratory investigation programmes made it not feasible and inefficient to run a full metabolic investigation programme in every new patient suspected of inherited metabolic disease. An application form for metabolic investigation is described, which can be used to collect clinical information relevant to metabolic disease. On the basis of the patient's clinical information, selection criteria are given to decide which laboratory investigation programme has to be performed in the individual patient. A full metabolic laboratory investigation programme is described and illustrated with some examples of abnormal metabolite patterns. Diagnostic results over a 2-year period are presented.