[From gene to disease: cystinosis]. / Van gen naar ziekte; cystinose.

Cystinosis is an autosomal recessive disorder caused by an impaired transport of cystine out of lysosomes. The most severe infantile form of cystinosis starts with Fanconi syndrome at the age of 3-6 months. Untreated patients develop renal failure before the age of 10. The cystinosis gene (CTNS) maps to chromosome 17p13, spans 23 kb and is composed of 12 exons. CTNS encodes a 367 amino acid protein, cystinosin, which is a H(+)-driven lysosomal cystine transporter. It is enigmatic how lysosomal cystine accumulation induces the clinical symptoms. ATP depletion was demonstrated in an experimental model consisting of loading lysosomes with cystine dimethylester. The amino-thiol cysteamine depletes lysosomal cystine content by a disulfide-exchange reaction with cystine. Therapy with cysteamine should be administered as early as possible and continued after a renal transplantation as the extra renal damage still progresses. Improved life expectancy of cystinotic patients requires the attention of internists with a special interest for this rare disorder.

Negligible urinary cysteamine loss in cystinosis patients with Fanconi syndrome.

Cystinosis is an inborn error of lysosomal cystine transporter, resulting in cystine accumulation in lysosomes of all cells. Renal Fanconi syndrome is an early sign of kidney involvement in cystinosis patients. Cysteamine, a small amino-thiol, depletes intralysosomal cystine content and reduces organ damage. However, it does not reverse renal Fanconi syndrome and only postpones the progression to renal failure. We examined whether cysteamine could be lost in the urine of cystinosis patients with Fanconi syndrome, which may explain the inefficiency of treatment. Urinary cysteamine loss was studied in 6 cystinosis patients with and without Fanconi syndrome and was less than I% of ingested dose in all patients.

The homocysteine distribution: (mis)judging the burden.

The nonfasting plasma total homocysteine (P-tHcy) concentration was measured in a random sample of 3025 Dutch adults aged 20-65 years (main study). The positively skewed distribution had a geometric mean of 13.9 micromol/L in men and 12.6 micromol/L in women. Blood of the main study was not cooled or centrifuged immediately after drawing. A stability study (n = 26) indicated that this could have resulted in a small (0.4 micromol/L) overestimation of the means. A comparative study (n = 88), and a reproduction of these results in an entirely different population (n = 213), showed a systematic difference in P-tHcy concentration of -2.4 micromol/L between our laboratory (Nijmegen, the Netherlands) and that in Bergen, Norway. With the information of the additional studies we provided precise and valid data of the Dutch P-tHcy distribution, from which we conclude the status in the Netherlands is worse than in other European countries. Furthermore, we showed that comparison of P-tHcy data is complicated unless the interlaboratory differences are known. @ 2001 Elsevier Science Inc.

New method for determining cystine in leukocytes and fibroblasts.

BACKGROUND: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. METHODS: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. RESULTS: The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.

Altered folate and vitamin B12 metabolism in families with spina bifida offspring.

Folic acid intake reduces the risk of neural tube defects (NTDs). Although the 677C-->T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene is a risk factor for NTDs, it only partly explains the elevated homocysteine levels in mothers of children with NTDs. We measured vitamin B12, folate and homocysteine in patients with spina bifida (SB), their parents, and in controls, to investigate which other enzymes of homocysteine metabolism might be defective. Because homozygosity for the 677C-->T mutation causes decreased plasma folate and increased red-cell folate (RCF) and plasma homocysteine levels, we excluded individuals homozygous for that mutation. The remaining SB patients and their parents still had lowered plasma folate and elevated total homocysteine levels, and a small subset had decreased vitamin B12 levels. Red-cell folate was the same in all groups, suggesting that dietary folate intake and its uptake was normal. Risk of SB was increased at the 25th percentile of plasma folate and at the 75th percentile of homocysteine values in SB patients and their parents, and at the 5th and 25th percentiles of vitamin B12 in mothers with SB-affected offspring. This underlines the functional importance of homocysteine remethylation to methionine. There was no correlation between vitamin B12 and homocysteine or RCF. In combination with the lowered plasma folate (80-90% 5-methyltetrahydrofolate), our data do not support a major involvement of methionine synthase in the aetiology of SB. Our data rather favour the involvement of genetic variation at loci coding for the formation of 5-methyltetrahydrofolate, such as MTHFR, methylenetetrahydrofolate dehydrogenase or serine hydroxymethyltransferase.

Decrease in S-adenosylmethionine synthesis by 6-mercaptopurine and methylmercaptopurine ribonucleoside in Molt F4 human malignant lymphoblasts.

6-Mercaptopurine (6-MP) and methylmercaptopurine ribonucleoside (Me-MPR) are purine anti-metabolites which are both metabolized to methylthio-IMP (Me-tIMP), a strong inhibitor of purine synthesis de novo. Me-MPR is converted directly into Me-tIMP by adenosine kinase. 6-MP is converted into tIMP, and thereafter it is methylated to Me-tIMP by thiopurine methyltransferase, an S-adenosylmethionine (S-Ado-Met)-dependent conversion. S-Ado-Met is formed from methionine and ATP by methionine adenosyltransferase, and is a universal methyl donor, involved in methylation of several macromolecules, e.g. DNA and RNA. Therefore, depletion of S-Ado-Met could result in an altered methylation state of these macromolecules, thereby affecting their functionality, leading to dysregulation of cellular processes and cytotoxicity. In this study the effects of 6-MP and Me-MPR on S-Ado-Met, S-adenosylhomocysteine (S-Ado-Hcy), homocysteine and methionine concentrations are determined. Both drugs cause a decrease in intracellular S-Ado-Met concentrations and an increase in S-Ado-Hcy and methionine concentrations in Molt F4 human malignant lymphoblasts. The effects of both 6-MP and Me-MPR can be ascribed to a decreased conversion of methionine into S-Ado-Met, due to the ATP depletion induced by the inhibition of purine synthesis de novo by Me-tIMP. Both 6-MP and Me-MPR thus affect the methylation state of the cells, and this may result in dysregulation of cellular processes and may be an additional mechanism of cytotoxicity for 6-MP and Me-MPR.

Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.