Determination of the lysosomal hydrolase activity in blood collected on filter paper, an alternative to screen high risk populations.

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.

Novel mutations in the glucocerebrosidase gene of brazilian patients with Gaucher disease.

Gaucher disease (GD) is an autosomal recessive disorder resulting from glucocerebrosidase (GC) deficiency due to mutations in the gene (GBA) coding for this enzyme. We have developed a strategy for analyzing the entire GBA coding region and applied this strategy to 48 unrelated Brazilian patients with GD. We used long-range PCR, genotyping based on the Taqman® assay, nested PCR, and direct DNA sequencing to define changes in the gene. We report here seven novel mutations that are likely to be harmful: S125N (c.491G>A), F213L (c.756T>G), P245T (c.850C>A), W378C (c.1251G>C), D399H (c.1312G>C), 982-983insTGC (c.980_982dupTGC), and IVS10+1G>T (c.1505+1G>T). The last alteration was found as a complex allele together with a L461P mutation. We also identified 24 different mutations previously reported by others. G377S was the third most frequent mutation among the patients included in this study, after N370S and L444P. Therefore, this mutation needs be included in preliminary screens of Brazilian GD patients. The identification of mutant GBA alleles is crucial for increasing knowledge of the GBA mutation spectrum and for better understanding of the molecular basis of GD.

Influence of pre-analytical factors on α-galactosidase A, arylsulfatase B and α-glucosidase activities measured on dried blood spots on filter paper.

OBJECTIVES: To analyze the effect of blood collection and storage conditions on activity of α-galactosidase A, arylsulfatase B and α-glucosidase. DESIGN AND METHODS: Blood was collected in EDTA, heparin, or direct spotting on filter paper and stored at different temperatures (-20, 4, 25 and 37°C) and storage times (3, 10, 17 and 180 days). The influence of filter paper size was also assessed (3.0 and 1.2mm). RESULTS: No statistically significant difference was observed between the three collection methods. α-Glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used. CONCLUSIONS: Both paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection.

Effect of sample collection, temperature and time of storage on ß-galactosidase and total hexosaminidase activities in dried blood collected on filter paper.

BACKGROUND: Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. METHODS: In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for ß-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. RESULTS: Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of ß-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. CONCLUSIONS: The two enzymes investigated in the present study may be stored for up to 17 days (ß-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.

Twelve different enzyme assays on dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases.

BACKGROUND: Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays. METHODS: Fluorometric methods were used to evaluate the activities of arylsulfatase B, alpha-N-acetylglucosaminidase, chitotriosidase, alpha and beta-galactosidases, beta-glucosidase, beta-glucuronidase, total hexosaminidases, hexosaminidase A, alpha-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 degrees C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured. RESULTS: Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays. CONCLUSIONS: Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.

Application of a comprehensive protocol for the identification of Gaucher disease in Brazil.

Gaucher disease (GD) is a sphingolipidosis caused by a genetic defect that leads to glucocerebrosidase (beta-glucosidase) deficiency. Between January 1982 and October 2003, 1,081 blood samples from patients suspected of having GD were referred for biochemical analysis. The activities of the enzymes beta-glucosidase (beta-glu) and chitotriosidase (CT) were measured in these samples. Among the 412 diagnosed cases of GD (38.1%), the great majority were GD type 1. The Brazilian regions with the greatest concentration of these patients were the Southeast, South, and Northeast. The mean age of patients at diagnosis was 19 years. The activity of beta-glu in patients with GD was, on average, 10.7% of that of normal individuals. CT was, on average, 269 times more elevated in this group of patients. Among the 669 cases with no confirmation of GD, there were patients with Niemann-Pick disease types A, B, or C (44 cases), possible heterozygotes for GD (59 cases), patients with other lysosomal storage diseases (LSDs) (19 cases) or with other inborn errors of metabolism (3 cases). In 508 cases, no metabolic disorder was found. This study shows that the biochemical protocol employed was effective for the detection of GD, a disease that is reasonably frequent in Brazil.

Increased triglyceride levels in a Japanese population living in southern Brazil.

BACKGROUND: In the present study we investigated 96 individuals of Japanese descent living in southern Brazil (Cascavel-PR) in terms of triglyceride (TG) levels (> or < or = 200 mg/dL) and compared them to non-Japanese control individuals. METHODS: We analyzed TG and total cholesterol (TC) levels by an enzymatic method and apolipoprotein A-I and B (apo A-I and apo B) by a turbidimetric method. We also determined the lipoproteins HDL and LDL by a direct method and by electrophoresis. All these determinations were performed in plasma. RESULTS: TG levels were above 200 mg/dL in 18.7% of the individuals of Japanese descent and in 8.4% of the controls. Mean TC levels were 259 mg/dL for Japanese descendants and 225 mg/dL for the control group. We observed that individuals of Japanese descent with TG levels above 200 mg/dL had the highest TC, LDL-c, and VLDL-c levels and the lowest HDL-c and apo A-I levels. Body mass index (BMI) was also higher in individuals of Japanese descent with TG above 200 mg/dL. CONCLUSIONS: This Japanese population has high TG levels compared to control individuals, and diet did not influence these levels.

G(M1)-ganglioside degradation and biosynthesis in human and murine G(M1)-gangliosidosis.

BACKGROUND: Gangliosides are building blocks of cell membranes and their biosynthesis and degradation have been extensively studied in the past. Regulation of the metabolism of these glycolipids controls fundamental cell functions. G(M1)-gangliosidosis, a neurodegenerative glycosphingolipid storage disease, is caused by deficiency of lysosomal beta-galactosidase with consequent disruption of the normal degradative pathway of G(M1)-ganglioside. We studied the impact of G(M1)-ganglioside accumulation on its biosynthetic enzyme in cells and tissues from human patients and from the G(M1)-gangliosidosis mouse model. METHODS: We tested the qualitative and quantitative pattern of gangliosides by thin layer chromatography and N-acetylneuraminic acid dosage, respectively. Regulation of G(M1)-ganglioside biosynthesis was evaluated by G(M1) synthase assay in human and murine samples. RESULTS: G(M1)-ganglioside accumulation has an inhibitory effect on the human but not on the mouse G(M1) synthase. We present evidence that G(M1) synthase activity in human and murine cells are regulated by different mechanisms. CONCLUSIONS: Alternative pathways in the mouse may account for these results and possibly explain some of the phenotypical differences between the human and mouse forms of this disorder.

Biochemical characterization of chitotriosidase enzyme: comparison between normal individuals and patients with Gaucher and with Niemann-Pick diseases.

OBJECTIVES: The aim of the present study was to establish the range of chitotriosidase (CT) activity in normal individuals, patients with Gaucher disease (GD) and Niemann-Pick disease (NPD), types A or B. The kinetics of CT in these three groups was also investigated. DESIGN AND METHODS: CT activity, as well as Km, Vmax, optimum pH, and thermal stability of the enzyme were determined in the plasma of control, GD, and NPD subjects. RESULTS: CT activity in GD and NPD patients was, respectively, around 600-fold and 30-fold greater than in normal individuals. We observed significant differences in optimum pH, Vmax, and thermal stability between the various groups. Km was different in normal individuals relative to GD and NPD patients. However, there was no significant difference between Km values in patients with GD and with NPD. CONCLUSIONS: Based on the differences found in the biochemical parameters studied, our results may be important to help the identification of patients not only with GD but also with NPD.

Investigation of lysosomal storage diseases in nonimmune hydrops fetalis.

OBJECTIVE: To investigate lysosomal storage diseases (LSD) in cases of nonimmune hydrops fetalis (NIHF). METHODS: Thirty-three cases of NIHF were investigated, 28 in the prenatal period and 5 in hydropic newborns. In addition to a general investigation for NIHF, specific enzymatic analyses for the detection of LSD were performed. RESULTS: In our sample, we detected five patients (15%) with LSD, each patient having one of the following diseases: mucolipidosis, Niemann-Pick disease, galactosialidosis, sialidosis and mucopolysaccharidosis type IV A. CONCLUSION: Although LSDs are rare disorders as a group, they should be considered as a possible cause of NIHF, even in the absence of consanguinity or of a previous family history. By excluding the more frequent causes of NIHF, an LSD investigation assists in clarifying the etiology of many hydropic cases, making more appropriate genetic counseling for parents possible.

Biochemical study on beta-glucosidase in individuals with Gaucher's disease and normal subjects.

BACKGROUND: Gaucher's disease (GD) is a disorder caused by the deficiency of lysosomal beta-glucosidase, an enzyme that participates in the degradation of glycosphingolipids. Deficiency of this enzyme results in the accumulation of glucocerebrosides in macrophage lysosomes. No studies comparing the biochemical and kinetic behavior of this enzyme in leukocytes and fibroblasts from normal individuals and patients with Gaucher's disease are available. METHODS: We compared the activities of beta-glu and chitotriosidase between normal subjects and Gaucher disease patients, and characterized the behavior of beta-glu in terms of pH optimum, heat stability, Km and Vmax. RESULTS: The results showed a different behavior of the enzyme in the groups analyzed. CONCLUSIONS: This finding might be useful in cases in which the measurement of enzyme activity alone is not reliable for the establishment of the diagnosis of Gaucher's disease.

Effect of a peroxysomal proliferator agent on intracellular cholesterol accumulation in cultured fibroblasts from Niemann-Pick type C disease patients.

BACKGROUND: Niemann-Pick type C (NP-C) disease is a lysosomal storage disorder. It is possible that peroxisomes are also modified and their alterations can be an early event in the process of the disease. As the use of peroxisomal inducers restores the original function of the organelle, the importance of peroxisomes is further emphasized and can suggest future therapeutic interventions. METHODS: We treated fibroblast cultures from NP-C patients and normal individuals with 200 and 400 micromol/l clofibrate and evaluated its action on intracellular cholesterol content that was determined by filipin staining and quantitative measurement of unesterified cholesterol. RESULTS: The fibroblasts from NP-C patients that did not receive any drug presented a pattern of intense perinuclear fluorescence associated with the accumulation of unesterified cholesterol which was not observed in normal fibroblasts. Comparing the NP-C fibroblasts that were incubated with clofibrate and the same cells without this treatment, there were no changes in cholesterol content by filipin staining, but normal fibroblasts after incubation with this drug showed a slight increase in its cholesterol content. However, unesterified cholesterol was significantly increased in both cells treated with clofibrate when compared to untreated cells. CONCLUSIONS: Clofibrate is probably not useful for treatment of NP-C patients because it seems to contribute to an increase the cholesterol in the cells of these individuals.

Application of a protocol for the detection of disorders of sialic acid metabolism to 124 high-risk Brazilian patients.

Lysosomal storage disorders (LSD) present great clinical variability. Included in this group are sialic acid metabolism disorders (SAMD). In the present study, we describe the application of a 3-step protocol for the diagnosis of SAMD, including (1). oligosaccharide and sialyloligosaccharide chromatography; (2). quantitative determination of sialic acid; and (3). measurement of neuraminidase activity. Application of our protocol to 124 individuals at risk for SAMD led to the diagnosis of five affected patients, two with type I sialidosis, one with type II sialidosis, and two with galactosialidosis. Due to its simplicity and efficiency, we propose the use of this protocol for the diagnostic evaluation of patients with suspected SAMD, which could be specially useful to non-specialized laboratories and to services located in developing countries.

Detection of organic acidemias in Brazil.

BACKGROUND: Organic acidurias or organic acidemias are inherited metabolic disorders in which organic acids (carboxylic acids) accumulate in tissues and physiologic fluids of affected individuals. They are considered the most frequent metabolic disorders among severely ill children. Patients frequently present acute symptoms in early life. Metabolic acidosis and neurologic symptoms are the most common signs. METHODS: Urine specimens obtained from 1,926 children from January 1994 to July 2001 were used in analyses. Venous blood specimens were also collected from some patients. Samples were initially submitted to screening tests for detection of inborn errors of metabolism. Identification and semi-quantitation of organic acids in urine were performed by gas chromatography or gas chromatography coupled to mass spectrometry using capillary column (DB-5) and flame ionization detection. RESULTS: Ninety three (4.8%) cases of organic acidemias were diagnosed among 1,926 patients investigated from January 1994 to July 2001. Prompt therapy was instituted after diagnosis in a considerable number of patients and resulted in rapid improvement in their symptomatology, distinct from our previous cases diagnosed abroad where patients representing index cases died before any measure could be taken. CONCLUSIONS: Results demonstrate the importance of diagnosing organic acidurias in loco in developing countries despite implied extra costs.

Effect of CuCl2, NaCl and EDTA on the enzyme alpha-L-iduronidase in the plasma of normal individuals and heterozygotes for MPS I.

BACKGROUND: It has been previously demonstrated that the enzyme alpha-L-iduronidase (IDUA) of patients with MPS I shows a different biochemical behavior in each of the three clinical forms of these. In heterozygotes, its biochemical behavior has been recently established in leukocyte and plasma samples, demonstrating that it is possible to distinguish individuals heterozygous for MPS I within an unselected population. METHODS: We evaluated the effect of copper chloride, EDTA and sodium chloride on the activity of the enzyme alpha-L-iduronidase in the plasma of normal individuals and of MPS I heterozygotes and observed the type of inhibition caused, the Ki, the apparent Km and the apparent Vmax for each inhibitor. RESULTS: Sodium chloride inhibited the enzyme in normal individuals and in 40% of the heterozygotes evaluated and activated it in 60% of heterozygotes. The remaining compounds inhibited IDUA in both heterozygotes and normal individuals. CONCLUSIONS: We detected significant differences capable of differentiating MPS I heterozygotes from normal individuals by simply adding sodium chloride, EDTA or copper chloride to the incubation medium at the time of IDUA activity determination, with a potential use in carrier detection protocols.

Detection of mucopolysaccharidosis type I heterozygotes based on the biochemical characteristics of leukocyte alpha-L-iduronidase.

BACKGROUND: In the present study, we biochemically characterized the enzyme alpha-L-iduronidase (IDUA) of leukocytes from normal individuals and from mucopolysaccharidosis I (MPS I) heterozygotes, and compared these characteristics to discriminate for inclusion into two different groups. METHODS: We fluorimetrically measured IDUA activity in leukocytes using 4-methylumbelliferyl-alpha-L-iduronide as an artificial substrate. Optimum pH, Km, Vmax, and thermostability of the enzyme at 50 degrees C were determined. RESULTS: Based on leukocyte IDUA activity, we divided the heterozygotes into two groups, one (group 1) with activity below that detected in controls, and the other with activity similar to that of normal individuals (group 2). The optimum pH for IDUA was 2.7 for normal individuals and 2.6-2.8 for heterozygotes. With respect to Km, there was a difference only between the value for normal IDUA (0.60 mM) and the value for group 2 (0.38 mM), while group 1 showed a statistically similar value (0.49 mM). The Vmax of the reaction was discriminated in the three groups in a highly effective manner. The IDUA of normal individuals had a higher Vmax (60.98 nmoL/h x mg protein) than the enzyme of group 1 heterozygotes (28.66 nmoL/h x mg protein) and the enzyme of group 2 (31.78 nmoL/h x mg protein). When the IDUA from the three groups was pre-incubated at 50 degrees C, we observed that the IDUA of both group 1 and group 2 was significantly more thermostable than the IDUA of normal individuals. CONCLUSIONS: Determination of IDUA activity alone is not sufficient to discriminate between MPS I heterozygotes and normal individuals because a considerable overlap occurs between them. Our study showed that leukocyte IDUA from MPS I heterozygotes differed from the normal enzyme in terms of optimum pH, Km, and Vmax of the reaction and thermostability at 50 degrees C. These parameters provide a simple and reliable tool for the detection of carriers for MPS I.

Detection of metabolic disorders in high-risk patients: a pilot study in Salvador, Bahia

The purpose of this pilot-study was to evaluate the applicability of a screening protocol for the detection of inborn errors of metabolism (IEM) in high-risk patients. The protocol was applied in 65 patients referred to the Medical Genetics Laboratory of the University Hospital Professor Edgard Santos due to the suspicion of an IEM. Eight of these patients (12.3 percent) displayed an abnormal result in the screening protocol. These patients, along with 22 who displayed normal results in the screening protocol but who presented clinical symptoms or signs suggestive of an IEM not detectable by the tests applied, were selected for a further diagnostic investigation. In 5 of these 30 patients (7.7 percent of the total sample) it was possible to establish the diagnosis of an specific IEM. The results indicate that the designed screening protocol was sucessfully applied, allowing the detection of affected patients in a frequency comparable to that observed in larger studies performed elsewhere. The continuation of this study and the enlargement of the sample will help to delineate the profile of IEM in northeast of Brazil and will allow the identification of a significative number of patients and families. who could benefit from the therapeutic and preventive measures available for these diseases.