Possible genetic defects in regulation of glycosaminoglycans in patients with diabetic nephropathy.

The hypothesis of genetic defects in glycosaminoglycan (GAG) regulation among patients with insulin-dependent diabetes mellitus (IDDM) and nephropathy was assessed by studies in tissue cultures of fibroblasts obtained from 7 patients with normal urinary albumin excretion, 11 patients with diabetic nephropathy, and 6 nondiabetic control subjects. The incorporation of [2H] glucosamine and [35S] sulfate into hyaluronic acid (HA), chondroitin sulfate and dermatan sulfate (CS + DS), and heparan sulfate (HS) was measured in cells, matrix, and medium and related to micrograms of tissue protein. Large interindividual variations were seen in all three groups, and the incorporation of [3H] glucosamine into HA, CS + DS, and HS and [35S] sulfate into CS + DS and HS were not significantly different between the three groups. However, the fractional incorporation of [3H]glucosamine into HS was significantly reduced in diabetic patients with nephropathy compared with control subjects. This was the case not only when related to the total amount of GAGs (P = 0.014) but also when related to HA (P = 0.014). No significant difference was seen between control subjects and normoalbuminuric diabetic patients. The degree of N-sulfation of HS was not significantly different between the experimental groups. The results suggest that patients with diabetic nephropathy may suffer from deficiencies of coordinate regulation in the biosynthesis of GAG in fibroblasts, which may lead to a reduced density of HS in the extracellular matrix. If these changes reflect alterations in the biosynthesis of GAG from endothelial, myomedial, and mesangial cells, this observation may be relevant for the pathogenesis of severe diabetic complications.

Tetrahydrobiopterin and Parkinson's disease.

Two patients with Parkinson's disease were treated with 1 g tetrahydrobiopterin (BH4) for 5 days. Clinical improvement was not observed. In the cerebrospinal fluid (CSF) a 4-8 fold increase in the concentration of homovanillic acid (HVA), and a 3-fold increase in the concentration of 5-hydroxyindole acetic acid (5-HIAA) was measured. However, the concentration of HVA reached, was only approximately half as high, as that of patients treated with madopar (DOPA + benserazid). In urine, the excretion of HVA increased 13-37 fold, when the patients were treated with madopar, whereas no increase in the HVA excretion was measured after the BH4 administration. Additionally, 2 patients with Parkinson's disease were treated with 1 g BH4 in combination with 15 g tyrosine for 3 days, and 1 parkinsonian patient was treated with 15 g tyrosine daily for 7 weeks. No increase in the CSF concentrations of HVA or 5-HIAA was observed. The results suggest, the BH4 in the dosage used, is not effective in the treatment of Parkinson's disease.

Increased neurotransmitter biosynthesis in phenylketonuria induced by phenylalanine restriction or by supplementation of unrestricted diet with large amounts of tyrosine.

Seven phenylketonuria (PKU) patients aged 15-24 years were allowed unrestricted diet for 3 weeks. Three of these patients performed well on unrestricted diet according to visual reaction time variability (RTv 50-100 ms) and did not show significant changes when returning to the phenylalanine-restricted diet (RTv 70-100 ms). Neither did the concentrations of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) change significantly. Four of the patients, however, performed rather poorly (RTv 120-220 ms) on unrestricted diet and improved significantly (P less than 0.03) when the diet was restored (RTv 70-150 ms). The improvements were accompanied by significant (P less than 0.01 and P less than 0.02) increases (mean 52% and 109%) in CSF levels of HVA and 5-HIAA. Five PKU patients aged 15-23 years were allowed unrestricted diet or unrestricted diet supplemented with various amounts of tyrosine (106-194 mg/kg per 24 h). Two of these patients performed very well on unrestricted diet (RTv 60 ms) and showed little change when the unrestricted diet was supplemented with tyrosine (RTv 70 ms and 80 ms). The three other patients, who performed rather poorly (RTv 120-220 ms), improved significantly (P less than 0.03) when the unrestricted diet was supplemented with tyrosine (RTv 70-140 ms). HVA in CSF increased significantly (P less than 0.01) with the tyrosine supplement when the amount exceeded a threshold of approximately 80 mg/kg per 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased vigilance and dopamine synthesis by large doses of tyrosine or phenylalanine restriction in phenylketonuria.

In a group of 9 patients with classical phenylketonuria (PKU) aged 15-24 years we examined the effect of phenylalanine restricted diet on vigilance, as judged by the continuous visual reaction times, and neurotransmitter synthesis, as judged by cerebrospinal fluid (CSF) homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) levels. HVA and 5-HIAA levels decreased significantly with increase in plasma phenylalanine concentration on free diet (p less than 0.01 and p less than 0.0005 respectively). Vigilance improved on phenylalanine restricted diet in 6 of the 7 patients with abnormally long reaction times on free diet. Addition of tyrosine (160 mg/kg/24 h) to the free diet resulted in an increased CSF in the six patients examined. In 14 patients on free diet supplemented with tyrosine, an improvement in vigilance (reaction times at the 90 percentile) was seen in all 12 patients with values longer than the normal mean (264 msec) (p less than 0.001). Tyrosine treatment may be a therapeutical alternative when phenylalanine restriction is impractical.

Normal excretion of urinary acid mucopolysaccharides in a boy with iduronate sulphatase deficiency, Hunter phenotype and alpha 1-antitrypsin deficiency.

The chance coincidence of an X-linked disorder with an autosomal recessive disorder in one child is described. The child had the clinical phenotype of a mucopolysaccharidosis and the activity of iduronate sulphatase was almost absent. Furthermore, fibroblasts from a typical Hunter patient were unable to correct the patient's fibroblasts. However, three 24 h urine samples collected at 18-36 months of age showed a nearly normal excretion of acid mucopolysaccharides. The boy died in liver coma at 3 years of age. Autopsy showed cirrhosis of the liver and changes in liver tissue consistent with alpha 1-antitrypsin deficiency.

Decreased vigilance and neurotransmitter synthesis after discontinuation of dietary treatment for phenylketonuria in adolescents.

Four adolescent or young adult patients with phenylketonuria were examined before and after discontinuation of dietary treatment. Plasma and CSF phenylalanine concentrations increased about two-fold in three patients. In these patients the CSF concentration of the dopamine and serotonin metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) decreased markedly; 5-HIAA to extremely low values. The reaction time variability increased in these patients. In the fourth case plasma phenylalanine levels, CSF HVA and 5-HIAA levels, and reaction time variability were essentially unchanged. The relationship between reaction time variability and the CSF 5-HIAA level for all four patients could be presented as a linear function. However, a causal relationship is still unproven. These preliminary findings demonstrate that there may be hazards in the discontinuation of dietary treatment, even in adolescents or young adults, for neurotransmitter metabolism and mental function.

Combined tetrahydrobiopterin-phenylalanine loading test in the detection of partially defective biopterin synthesis.

Deficiency in the synthesis of biopterin causes neonatal hyperphenylalaninemia. We report a 10-year-old girl of normal appearance with a partial defect in biopterin synthesis, normal intelligence and normal serum phenylalanine levels (95 mumol/l) (1.6 mg/dl). During her 1st year of life serum phenylalanine levels were 250 mumol/l (4 mg/dl) and phenylalanine loading performed at 6 months and 1 year of age was not followed by an increase in serum tyrosine. At 9 years of age she had developed a severely abnormal EEG with focal spike activity but no observable clinical abnormalities. Determination of urinary pterins showed abnormal low levels of biopterin and high levels of neopterin. Phenylalanine loading combined with oral administration of tetrahydrobiopterin (BH4) was followed by a normal increase in serum tyrosine and a normal decrease in serum phenylalanine. Considering the importance of BH4 for the synthesis of dopamine, catecholamines, and serotonin we suggest that these cases should be followed carefully. If neurological symptoms appear, e.g., epilepsy, it may be worthwhile to consider treatment with BH4 and neurotransmitter precursors.

Metachromatic leukodystrophy and pseudoarylsulfatase A deficiency in a Danish family.

A child with a diagnosis of late-infantile metachromatic leukodystrophy (MLD), and a normal father with low arylsulfatase A (ASA) activity in leucocytes and cultured fibroblasts is described. The child had a pathologically increased amount of sulfatides in the urine, whereas no sulfatides could be found in the father's urine. Sulfatide-loading of the child's cultured fibroblasts showed an accumulation of sulfatides, whereas the fibroblasts from the father had a marginally decreased sulfatide turnover. It is thus possible to discriminate between these two forms of low ASA activity in this family, and to ensure a correct diagnosis should the amniotic fluid cells show a low ASA activity in future pregnancies.

Reliability of the use of fructose 1-phosphate to detect Hunter cells in fibroblast-cultures of obligate carriers of the Hunter syndrome.

Pulse-chase experiments measuring 35S-sulphate incorporation into acid mucopolysaccharides were performed in the presence and absence of fructose 1-phosphate on fibroblasts obtained from one skin-biopsy of 25 obligate Hunter carriers. The presence of fructose 1-phosphate significantly increased the accumulation of 35S-labelled acid mucopolysaccharides in fibroblast cultures of 23 obligate Hunter carriers. In one carrier, the accumulation of labelled acid mucopolysaccharides was significantly increased prior to the addition of fructose 1-phosphate, and in one of the 25 obligate carriers the 35S-sulphate incorporation was normal in the presence as well as in the absence of fructose 1-phosphate. Similar experiments performed on mixtures of Hunter cells and normal cells revealed that 20% Hunter cells should be present to obtain a significantly increased difference in between the incorporation in the presence and in the absence of fructose 1-phosphate. Fructose 1-phosphate had no effect on the accumulation of labelled mucopolysaccharides in fibroblast-cultures of seven women with no family history of mucopolysaccharidosis. The present results show that pulse-chase experiments measuring 35S-sulphate incorporation into fibroblasts, cultured in the presence of fructose 1-phosphate, can identify Hunter carriership, provided that the accumulation is normal prior to the addition of fructose 1-phosphate. Furthermore, 35S-sulphate incorporation in the absence of fructose 1-phosphate, higher than mean +4SD of normal control-fibroblasts indicates carriership.

Diagnosis of Hunter's syndrome carriers; radioactive sulphate incorporation into fibroblasts in the presence or fructose 1-phosphate.

Mutual correction of co-cultivated fibroblasts from patients with Hunter's and Hurler's syndrome could be inhibited by either fructose 1-phosphate or mannose 6-phosphate. In the presence of fructose 1-phosphate a 50% mixture of fibroblasts from a patient with Hunter's syndrome and a normal homozygous individual showed an increased 35S-sulphate incorporation into acid mucopolysaccharides. When fibroblast cultures from one obligate and two possible carriers of Hunter's syndrome were tested for 35S-sulphate incorporation, the cultures showed either twice the normal 35S-sulphate incorporation into acid mucopolysaccharides in the presence of fructose 1-phosphate or an abnormally high incorporation in the presence as well as in the absence of the sugar phosphate.

Detection of hyperlipidemia by semiquantitative fluorometric determinations of serum lipids on thin-layer chromatograms.

In one analytical procedure, semiquantitative information concerning the concentrations of triglycerides, free and esterified cholesterol were obtained. Twenty-five microlitres of serum were used and the concentrations were classified as high, borderline or normal compared to a lipid standard solution. Thirty-four patients were investigated and the results showed good agreement with the results from conventional screening for hyperlipidemia by separate quantitative analyses of total cholesterol and triglycerides.

Rapid micromethod for flourometric determination of neutral lipids on thin-layer chromatograms.

A rapid fluorometric procedure for in situ evaluation of neutral lipids on thin-layer chromatograms is described. The lipids are rendered fluorescent by treatment with perchloric acid, and the intensity of the emitted light is recorded by flying spot scanning. Detection limits (2.8 x SD) are 0.10, 0.11, and 0.17 nmol for triglycerides, cholesterol, and cholesterol esters, respectively. Range of linearity is 0--3.4 nmol for triglycerides, 0--2.0 nmol for cholesterol, and 0--4.0 nmol for cholesterol esters. When standard in triplicate and 2 samples of each unknown are applied to the chromatogram, standard error of the mean is 2.7% for triglycerides, 1.7% for cholesterol, and 1.0% for cholesterol esters. The sensitivity of the method allows simultaneous determination of the three lipid classes in 5 microliter lipid extract representing 0.238 microliter human plasma.

Plasma glucagon responses to insulin-induced hypoglycaemia and arginine in normal and alloxan diabetic rats.

A decreased plasma glucagon response to insulin-induced hypoglycaemia was found in the normoglucagonaemic alloxan diabetic rat. 0.021-0.240 ng/ml was accepted as the range of normoglucagonaemia. The reduction of the glucagon response was not due to destruction of the alpha 2-cells by alloxan, since a normal response to arginine could be demonstrated. The decreased glucagon response to hypoglycaemia in the insulin deficient normoglucagonaemic alloxan diabetic rat suggests that this alpha 2-cell dysfunction may be caused by insulin lack.