The interaction of lead exposure and arylsulfatase A genotype affects sulfatide catabolism in human fibroblasts.

Lead exposure causes cognitive and behavioral deficits in some affected children. We propose that a contributing mechanism for the neurological damage is that lead induces critically low levels of arylsulfatase A (ASA) at sensitive stages of nervous system development. It is hypothesized that the combined effects of a single nucleotide polymorphism (SNP) in human ASA which results in reduced levels of the enzyme, and lead concentrations which decrease ASA activity culminate in cellular enzymic activity that is below a critical threshold required for the maintenance of normal nervous system function. Human fibroblasts grown in the presence of 20 microM lead acetate exhibit a more than 60% decrease of cellular ASA enzyme protein. Lead treatment of cells from individuals with the SNP(s) of pseudodeficient ASA, but not those from subjects with the normal gene, results in a significant decrease in ability of the cells to desulfate sulfatide, the substrate of ASA. The decrease in the degree of sulfatide catabolism is consistent with possible enhanced lead-induced neurobehavioral effects in individuals homozygous for the pseudodeficiency polymorphism(s) of ASA.

The R496H mutation of arylsulfatase A does not cause metachromatic leukodystrophy.

Deficiency of arylsulfatase A (ARSA) enzyme activity causes metachromatic leukodystrophy (MLD). A number of ARSA gene mutations responsible for MLD have been identified. Recently, the R496H mutation of ARSA was proposed to be a cause of MLD (Draghia et al., 1997). We have investigated the R496H mutation and found this mutation at a relatively high frequency in an African American population (f=0.09, n=61 subjects). The ARSA enzyme activity in subjects with and without the R496H mutation was determined and found to be normal. It is therefore concluded that the R496H mutation of ARSA does not negatively influence the activity of ARSA and is not a cause of MLD.

Association of long variants of the dopamine D4 receptor exon 3 repeat polymorphism with Parkinson's disease.

The dopamine D4 receptor (D4DR) has a highly polymorphic region in the third exon which has been associated with novelty seeking (NS) behavior. Due to the central position of dopamine and the documented low NS in Parkinson's disease (PD), the frequency of the exon 3 variants of D4DR in 95 PD patients and 47 controls was investigated. A significantly higher frequency of exon 3 alleles with six or more repeat units was found in the PD group (p = 0.039). This provides evidence that some forms of the highly polymorphic D4DR may represent a genetic susceptibility factor for PD.

Association of a serotonin transporter gene promoter polymorphism with harm avoidance behaviour in an elderly population.

A polymorphic 44-nucleotide insertion/deletion in the promoter region of the serotonin transporter gene (5-HTTLPR) has been shown to affect the level of expression of the serotonin transporter protein. An association between anxiety-related behavioural traits and the short form of the 5-HTTLPR has been reported. We determined the 5-HTTLPR genotype in genomic DNA samples from 84 subjects (47 Parkinson's disease patients and 37 controls) with a mean age of 67.4 years. The TPQ of Cloninger was used to obtain values for harm avoidance (HA), reward dependence and novelty seeking for all subjects. Analysis of variance showed a significant influence of the s-allele of the 5-HTTLPR on HA in both subject groups, with no significant interaction between diagnosis and genotype. Subjects with the l/l-genotype had significantly lower mean HA scores than the l/s subjects (P < 0.04) and s/s subjects (P < 0.003). A linear change in HA with genotype was observed, indicating a gene dose effect of the 5-HTTLPR s-allele on this personality dimension. Based on these findings it is suggested that there may be increased influence of the 5-HTTLPR short allele on anxiety-related traits during aging.

Determination of galactose and galactocerebroside using a galactose oxidase column and electrochemical detector.

A method has been developed to measure galactose and galactocerebroside using galactose oxidase immobilized on a solid resin. Galactose oxidase converts galactose and galactocerebroside to their corresponding aldehydes and hydrogen peroxide, the latter being electroactive and measurable by electrochemical detection using DC amperometric detection. The minimal detection limits of galactose and galactocerebroside were 1 and 2 microM, respectively. The linear response to galactose and galactocerebroside was to at least 300 microM. About 100 samples can be measured per hour using flow injection analysis. The activity of sulfatidase (cerebroside-3-sulfate-3-sulfohydrolase), which converts sulfatide (sulfogalactocerebroside) to galactocerebroside, was measured, and its inhibition by O-phospho-L-tyrosine was determined.

A novel arylsulfatase A protein variant and genotype in two patients with major depression.

A new, 'diffuse, multiple banding', electrophoretic variant of arylsulfatase A protein was found in two patients with major depression. Protein analyses showed that this variant and the normal enzyme differed in amino acid sequence and/or post-translational modifications unrelated to phosphate groups and oligomannose glycans. Analysis of the arylsulfatase A genes from a subject with the new variant identified three mutations; one gene had the two mutations associated with arylsulfatase A pseudodeficiency, and the other had a G to T transversion which changes a tryptophan to cysteine in the protein. These mutations result in an arylsulfatase A protein heteromer with diffuse electrophoretic banding. The possible association of these mutations with major depression is discussed.

Arylsulfatase A pseudodeficiency-associated mutations: population studies and identification of a novel haplotype.

Pseudodeficiency of arylsulfatase A is characterized by reduction of arylsulfatase A activity without neurodegeneration, making it an important complication when diagnosing metachromatic leukodystrophy. Two DNA substitutions are associated with arylsulfatase A pseudodeficiency. One, 1788A-->G, results in the loss of an N-glycosylated asparagine in the protein, and the second, 2723A-->G, removes the polyadenylation signal site of the mRNA. Previously, the polyadenylation signal site variant was observed only in the presence of the N-glycosylation site variant, although the latter has been reported to occur in the absence of the polyadenylation signal site variant. We investigated the frequencies of these alleles and their linkage disequilibrium in a number of populations and in psychiatric patients. While the N-glycosylation site variant had a high frequency in the Bantu-speaking people from Southern Africa (0.44), the San of Southern Africa (0.22), African Americans (0.37), and Cheyenne Indians (0.375), the polyadenylation signal site variant was absent in these groups. The mutated polyadenylation signal site was found only in the Caucasian groups surveyed. Two Caucasian sibs were identified with the pseudodeficiency polyadenylation signal site variant in the absence of the N-glycosylation site variant, indicating that linkage disequilibrium between the two polymorphisms is not perfect.

Association of alcoholism with the N-glycosylation polymorphism of pseudodeficient human arylsulfatase A.

The IIIa and IIIb electrophoretic variants of arylsulfatase A (EC 3.1.6.8) are 12 times more prevalent in alcoholic than in nonalcoholic populations. These variant enzymes, found in a subset of alcoholics, possess the pseudodeficient Asn350-Ser mutation of arylsulfatase A and, consequently, lack an N-linked glycan unit. These genetically determined variants of arylsulfatase A show reduced intracellular half-life, and cells from such individuals possess reduced enzymic activity. We propose that this polymorphism is an underlying genetic and biochemical factor contributing to the neuropathology and/or addiction pathway of this disease.

Structural characterization of variant forms of arylsulfatase A that associate with alcoholism.

Several electrophoretic forms of human platelet arylsulfatase A (ASA), including variant type IIIa and normal type IV(a), have been identified by nondenaturing polyacrylamide gel electrophoresis. An alcoholic population that we have analyzed is enriched in variant type IIIa compared with nonalcoholic psychiatric and normal controls. Individuals with the IIIa enzyme possess greatly reduced levels of ASA activity. To understand further the structural basis for the differences and their potential biological consequences, the nature of the ASA variant expressed by fibroblasts from different individuals was explored. The electrophoretic patterns of fibroblast ASA from the IIIa and IV(a) individuals differ in degree of phosphorylation. Furthermore, fibroblast ASA from IIIa individuals lacks an N-linked glycan found in ASA from IV(a) individuals. In addition, differences in peptide and/or posttranslational modification unrelated to the N-linked carbohydrate or phosphorylation exist between the fibroblast ASA from IIIa and IV(a) individuals. The finding that both fibroblasts and platelets exhibit related electrophoretic isoform patterns characteristic of the donor's ASA type allows for the use of fibroblasts to study the impact of ethanol on the metabolism of cells possessing different ASA types.

Arylsulfatase A: relationship of genotype to variant electrophoretic properties.

Previous work has shown that specific electrophoretic variants of arylsulfatase A occur more frequently among alcoholic patients than among psychiatric and normal controls. The present study sequenced the gene for two of these electrophoretic variants, IIIa and IIIb. Both contain an A-to-G transition corresponding to substitution of Asn350 by Ser, with the resulting loss of an N-glycosylation site. The difference in electrophoretic mobility of their gene products is due to a mutation in the IIIb gene resulting in the replacement of Arg496 by His. Evidence is presented that individuals possessing either of two other electrophoretic variants, Va and Vb, are heterozygous for a normal ASA allele and either a IIIa or IIIb allele, respectively. Thus, the relationship between the phenotype of the electrophoretic banding patterns, IIIa, IIIb, Va, and Vb, and their corresponding genotypes has been elucidated.

A method for rapid detection of arylsulfatase A pseudodeficiency mutations.

Pseudodeficiency of arylsulfatase A is a complicating factor in the determination of metachromatic leukodystrophy risk and carrier status. A method using polymerase chain reaction and restriction enzyme digestion to detect the presence of both the mutations that contribute to arylsulfatase A pseudodeficiency is described using DNA from blood or buccal cells. Application of this technique should facilitate determination of metachromatic leukodystrophy status and counseling in families where the pseudodeficiency allele is present.

Galactose biosensors using composite polymers to prevent interferences.

A biosensor using a composite polymer to prevent interferences was used in a flow injection analysis system for the detection of galactose in human plasma. The biosensor consisted of galactose oxidase immobilized on a platinized carbon electrode that had been modified with a composite polymer. The composite polymer showed improved selectivity to hydrogen peroxide compared with either of its individual polymeric components, Nafion and a copolymer of diaminobenzene and resorcinol. The composite polymer minimized the effect of possible interference from urate, ascorbate, and acetaminophen. This analytical system had a minimum detection limit of 50 microM, linearity to 6 mM, a storage stability of greater than 30 days, and a high sample throughput (approx. 120 samples/h).

Factors determining the severity of pathological gambling in males.

In extending the implications of our earlier research, we found that a measure of impulsivity developed by Barratt (1965) differentiated recovering, male pathological gamblers (N = 12; mean age = 48.9 years) from male control subjects (N = 15; mean age = 43.3 years). Among the gamblers themselves, however, this measure of impulsivity did not correlate with an index of the social and familial disruption engendered by past gambling. In contrast, a measure of one facet of the gamblers' cognitive style (the TF subscale of the Myers-Briggs Inventory) did correlate with this index of gambling-induced disruption but did not differentiate gamblers from controls. These results, as well as other findings, are discussed in the context of previous research and with regard to the inferential limits imposed by studies of this kind.

Uric acid level increases in humans engaged in gambling: a preliminary report.

The effect of gambling and gaming on plasma levels of uric acid was studied. Blood samples were obtained from normal subjects while they gambled for money or while they played checkers without betting. There was an interaction of time and activity reflecting primarily an association of increased uric acid levels during gambling over time, compared with gaming and relaxation. This indicates that gambling can increase plasma levels of uric acid.

Relation between serum uric acid and blood pressure in adolescents.

In adults, serum uric acid is positively associated with blood pressure levels. It is also a predictor of the development of hypertension in normotensive adults. The purpose of this study was to examine the relation of serum uric acid to systolic and diastolic blood pressure in adolescents. The data, from Cycle III of the National Health Examination Survey, consisted of a national probability sample of 6768 youths, 12-17 years old, in the United States. With age, height, weight, and sexual maturity controlled, serum uric acid significantly predicted blood pressure in adolescents. This relationship of uric acid and blood pressure was evident in male, but not female, adolescents. In association with findings from adult studies, these results indicate that uric acid levels may be useful indicators of adolescents at risk for hypertension.

MAST scores, alcohol consumption, and gynecological symptoms in endometriosis patients.

Alcohol consumption (quantity, frequency, and pattern) and alcohol-related problems were determined in endometriosis patients (n = 137), patients with other gynecological disorders (n = 91), and normal control subjects (n = 98). Participants completed a self-administered questionnaire, including the Michigan Alcoholism Screening Test (MAST), questions to determine the quantity and frequency of alcohol use, and questions regarding the relationship between gynecological symptoms and alcohol intake. The percentage of endometriosis patients with MAST scores greater than five or seven was significantly greater than that of normal control subjects (p = 0.045 and p = 0.009, respectively), but did not differ from that for patients with other gynecological disorders. Endometriosis patients with high MAST scores (> or = 5) tended to consume more alcohol on a yearly basis than normal control subjects with high MAST scores (p = 0.07). Among participants who experienced gynecological symptoms and were not abstainers, 31% of endometriosis patients, 9.5% of normal control subjects, and 14.3% of patients with other gynecological disorders reported increasing their alcohol consumption when experiencing gynecological symptoms. Endometriosis patients tended to differ in this regard from normal control subjects (p = 0.058) and were significantly different from patients with other gynecological disorders (p = 0.039). The evidence suggests that the gynecological problems of endometriosis may be a major medical correlative of alcoholism in women.

The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A.

Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure.

Behavioral restraint and symptoms of attention deficit disorder in alcoholics and pathological gamblers.

Adult alcoholics as well as pathological gamblers reported that, as children, they had higher than control levels of attention deficit disorder-related behaviors. On the other hand, alcoholics and only a subset of gamblers showed deficits in a test of behavioral restraint.

Electrochemical determination of arylsulfatase activity using high-performance liquid chromatography.

A highly sensitive assay for arylsulfatase A was developed using high-performance liquid chromatography with electrochemical detection. The retention time of the enzymatic product, p-nitrocatechol, on a reverse phase column was approximately 2.0 min. The assay was able to detect 0.43 pmol p-nitrocatechol in a 20-microliter ethanol extract of the reaction mixture. The coefficients of variation for seven determinations of the intra- and interassays were 9 and 8%, respectively. The levels of arylsulfatase A activity in human saliva samples and leukocyte and platelet lysates determined by this assay were within 6 and 11%, respectively, of the levels determined by a spectrophotometric assay. The high-performance liquid chromatography assay may have utility in measuring arylsulfatase A activity in biological samples with low activity or specific activity or in samples with compounds which interfere with the spectrophotometric assay.