Extracellular sulfatases as potential blood-based biomarkers for early detection of lung cancer.

PURPOSE: Non-small lung (NSCLC) is the deadliest cancer, with survival measured in months. Earlier diagnosis using a robust biomarker would likely improve survival. This study aims to determine whether blood levels of the extracellular sulfatases (SULF1 and SULF2) and their bio-activity can serve as novel biomarkers for NSCLC early detection. MATERIALS AND METHODS: Using human plasma specimens from NSCLC patients, nonmalignant COPD patients, and healthy individuals, we determined the association between plasma SULF levels and the presence of NSCLC. We assessed the plasma SULF levels as a function of sex and age. We also evaluated the plasma levels of heparin-binding factors potentially mobilized by the SULFs. To increase test specificity of blood SULF2 as a biomarker for the early diagnosis of NSCLC, we investigated the presence of a tumor-specific SULF2 isoform released in the blood, which could be used as a biomarker alone or in multiplex assays. RESULTS: The median level of plasma SULF2 was significantly elevated in NSCLC patients than in healthy controls (∼2 fold). However, these data were confounded by age. Surprisingly, COPD patients also showed a dramatically increased SULF2 plasma level. We showed a significant increase in the median plasma levels of several HSPG-binding factors in early-stage NSCLC patients compared to controls. Furthermore, we revealed a significant positive correlation of the SULF2 protein level with the plasma levels of two HSPG-binding factors IL6 and IL8. We demonstrated that NSCLC cancer cells and tissues overexpress a SULF2 splice variant. We determined the presence of a SULF2 splice variant form in NSCLC plasma, which was not detectable in COPD and control plasmas. CONCLUSION: Our findings highlight the potential for the plasma levels of SULF2 protein and its bio-activity as novel blood biomarkers for early diagnosis of NSCLC.

Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS.

Sulfonated steroids (s-St) have been usually regarded as inactive metabolites but are progressively considered as precursors for the intra-tissue formation of bioactive steroids. Moreover, independent effects without preceding removal of the sulfate group have been observed. We use the porcine testicular-epididymal compartment as a model to investigate the still largely unknown s-St physiology as the boar exhibits an intriguingly broad s-St spectrum predominantly originating from the testis. The application of LC-MS/MS in steroidomics enables the determination of unconjugated and intact sulfonated steroids with currently highest specificity and good sensitivity, allowing the concurrent measuring of numerous analytes in larger quantities of samples. Profiles (6h, 20min intervals) were generated for sulfonated 5-androstene-3ß,17ß-diol (Adiol-S), androsterone (A-S), dehydroepiandrosterone (DHEA-S), epiandrosterone (EA-S), epitestosterone (ET-S), estrone (E1-S), estradiol-17ß (E2-S), pregnenolone (P5-S), 17αOH-pregnenolone (OHP5-S) and unconjugated testosterone (T) in four unstimulated and four hCG-stimulated boars. Moreover, concentrations were measured in individual samples collected from testicular afferent and efferent blood to differentiate between testicular vs. extratesticular origin. Highest concentrations were found for EA-S, followed by ET-S, Adiol-S and DHEA-S, which mostly exceeded the levels of E1-S and A-S. Lowest concentrations were obtained for E2-S, P5-S and OHP5-S. The analytical profile also included sulfonated T, 5α-dihydrotestosterone and cholesterol. However, their concentrations were below the limit of quantification. Profiles of quantifiable s-St were consistent with a wave-like pattern associated with T pulses. In postpartal females (n=5) concentrations of all analytes assessed were undetectable, suggesting that in pigs the adrenals are not a quantitatively significant source of s-St.

Sulfatase modifying factor 1 (SUMF1) is associated with Chronic Obstructive Pulmonary Disease.

BACKGROUND: It has been observed that mice lacking the sulfatase modifying factor (Sumf1) developed an emphysema-like phenotype. However, it is unknown if SUMF1 may play a role in Chronic Obstructive Pulmonary Disease (COPD) in humans. The aim was to investigate if the expression and genetic regulation of SUMF1 differs between smokers with and without COPD. METHODS: SUMF1 mRNA was investigated in sputum cells and whole blood from controls and COPD patients (all current or former smokers). Expression quantitative trait loci (eQTL) analysis was used to investigate if single nucleotide polymorphisms (SNPs) in SUMF1 were significantly associated with SUMF1 expression. The association of SUMF1 SNPs with COPD was examined in a population based cohort, Lifelines. SUMF1 mRNA from sputum cells, lung tissue, and lung fibroblasts, as well as lung function parameters, were investigated in relation to genotype. RESULTS: Certain splice variants of SUMF1 showed a relatively high expression in lung tissue compared to many other tissues. SUMF1 Splice variant 2 and 3 showed lower levels in sputum cells from COPD patients as compared to controls. Twelve SNPs were found significant by eQTL analysis and overlapped with the array used for genotyping of Lifelines. We found alterations in mRNA expression in sputum cells and lung fibroblasts associated with SNP rs11915920 (top hit in eQTL), which validated the results of the lung tissue eQTL analysis. Of the twelve SNPs, two SNPs, rs793391 and rs308739, were found to be associated with COPD in Lifelines. The SNP rs793391 was also confirmed to be associated with lung function changes. CONCLUSIONS: We show that SUMF1 expression is affected in COPD patients compared to controls, and that SNPs in SUMF1 are associated with an increased risk of COPD. Certain COPD-associated SNPs have effects on either SUMF1 gene expression or on lung function. Collectively, this study shows that SUMF1 is associated with an increased risk of developing COPD.

Natural history of alpha mannosidosis a longitudinal study.

BACKGROUND: Alpha-Mannosidosis is a rare lysosomal storage disorder, caused by the deficiency of the enzyme alpha-Mannosidase. Clinically it is characterized by hearing impairment, skeletal and neurological abnormalities and mental retardation. In order to characterize the clinical features and disease progression of patients affected by alpha-Mannosidosis, a survey study was conducted. 43 patients from 4 European countries participated in this longitudinal study. Age range of the participants was 3 to 42 years. For each patient a medical history, complete physical and neurological examination, joint range of motion and assessment of physical endurance and of lung function were completed. In addition, serum and urinary oligosaccharide levels were analysed. METHODS: In this multicenter longitudinal study clinical data of 43 alpha-Mannosidosis patients were collected. In addition to objective clinical measurements biochemical assays were performed. RESULTS: Data analysis revealed a wide spectrum of clinical presentation regarding the severity and disease progression. Most clinical abnormalities were observed in the musculoskeletal and neurological system. All patients showed mental retardation and hearing loss from early childhood. An impairment in physical endurance was revealed by the 6-minute walk and 3-minute stair stair climb tests. There was only slight progression of a few clinical findings: Psychiatric troubles in both groups essentially, and respiratory dysfunction under 18 years. The serum and urinary oligosaccharide levels were increased in all affected individuals and correlated well with the 6-minute walk and 3-minute stair climb test results. CONCLUSIONS: This study confirms that alpha-Mannosidosis is a very heterogeneous disorder regarding both, disease severity and progression. As it has been shown that Mannosidosis patients are able to perform lung function tests and the 6MWT and stair-climb test, these clinical parameters apparently can be used as clinical endpoints for clinical trials. Oligosaccharide levels appeared correlated with functional testing and may serve as biomarkers of disease severity, progression and response to treatment. TRIAL REGISTRATION: ClinicalTrials.gov Identifier = NCT00498420 and EuropeanCommission FP VI contract LHSM-CT-2006-018692.

Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes.

Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis; however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration, and solid-phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated.

Development of an assay for the detection of mucopolysaccharidosis type VI patients using dried blood-spots.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disorder (LSD), which is caused by a deficiency in the enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). MPS VI is characterized by severe skeletal abnormalities, somatic tissue pathology and early death. Treatment possibilities include bone marrow transplantation (BMT) and enzyme replacement therapy (ERT; currently in phase III clinical trial). Early diagnosis of MPS VI will allow treatment before the onset of irreversible pathology. METHODS: Sensitive immune assays have been developed to detect 4-sulfatase protein and activity in normal control and MPS VI blood-spots. RESULTS: Dried blood-spots from MPS VI patients contained no detectable 4-sulfatase protein and activity, compared to 3.5-21 microg/L of 4-sulfatase protein and 291-1298 nmol/min/L of activity for normal human controls. In this evaluation study, the assay was sensitive and 100% specific, allowing reliable detection of individuals with MPS VI. CONCLUSIONS: The assays reported here have the potential to detect MPS VI patients using dried blood-spots.

Clinical, biochemical and molecular findings in a two-generation Morquio A family.

Mucopolysaccharidosis type IVA (Morquio A) is caused by a deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme capable of cleaving the sulfate group from both N-acetylgalactosamine-6-sulfate and galactose-6-sulfate. We describe here a two-generation Morquio A family with two distinct clinical phenotypes. The two probands from the second generation showed intermediate signs of the disease whereas their affected mother, aunt and two uncles had only very mild symptoms. Galactose-6-sulfatase (GALS) activity in leukocytes and fibroblasts of the affected family members was clearly deficient. Molecular genetic analysis of the GALNS gene revealed that two different point mutations segregate in the family, which correlated well with the clinical phenotype. The probands with intermediate symptoms were compound heterozygotes for the mutations R259Q and R94G, the latter one being inherited from the unaffected father. The mother and her affected siblings with the unusually mild phenotype were proven to be homozygous for the novel missense point mutation R259Q.

Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties.

Quercetin is one of the most abundant flavonoids in the human diet. This study aimed to determine the plasma concentrations of quercetin in 10 healthy volunteers after the consumption of a complex meal rich in plant products. Quercetin was determined in plasma (2 h before, and 3, 7 and 20 h after the meal), and in a duplicated portion of the meal by HPLC analysis with an electrochemical detection. The amount of ingested quercetin was estimated to be 87 mg. Before the meal, quercetin concentration in hydrolyzed plasmas ranged from 28 to 142 nM. A marked increase was observed 3 h after the meal in all subjects, with a mean concentration of 373 nM (S.E.M. = 61). After 7 h, quercetin concentration in hydrolyzed plasmas decreased and after 20 h basal levels were found again. The antioxidant capacities of quercetin, 3'-O-methylquercetin, and of some of their conjugated derivatives were compared by the measurement of the conjugated dienes resulting from the Cu2+-induced oxidation of human LDL. 3'-O-Methylquercetin and conjugated derivatives of quercetin significantly prolonged the lag phase, but the magnitude of their effect was about half that of the aglycone.

Measurement of oestrone sulphatase activity in white blood cells to monitor in vivo inhibition of steroid sulphatase activity by oestrone-3-O-sulphamate.

Formation of oestrone via the sulphatase pathway is considered to be a major source of the oestrogen present in breast tumours. Several inhibitors of steroid sulphatase have now been developed for use in the treatment of postmenopausal women with breast cancer. In order to be able to monitor the extent and duration of the inhibition of oestrone sulphatase (E1-STS) readily, we have developed a method to measure the activity of this enzyme in white blood cells (WBCs). Hydrolysis of oestrone sulphate by E1-STS in WBCs was linear with respect to time and the volume of WBCs used. To examine whether the extent of inhibition of E1-STS activity in WBCs, by the inhibitor oestrone-3-O-sulphamate (EMATE), reflected inhibition in other body tissues, activity in WBCs was compared with that in liver and spleen tissue samples from rats. Two hours after an oral dose of EMATE the extent of inhibition of E1-STS detected in WBCs was the same as in the liver. The duration of the inhibition of E1-STS by EMATE, examined over a 1-28 day period in rats, was similar whether monitored in WBCs, liver or spleen. Measurements of E1-STS activity in WBCs were also used to examine the effectiveness of EMATE (0.5 mg/kg) in two male volunteers. E1-STS activity was rapidly inhibited and had only recovered by 27% after 1 month. A marked decrease in the ratio of plasma dehydroepiandrosterone:dehydroepiandrosterone-sulphate (DHA:DHA-S) concentrations was also detected, confirming that EMATE also inhibits DHA-STS activity. The ability to monitor the extent and duration of steroid sulphatase inhibition in WBCs will facilitate the evaluation of this new form of endocrine therapy in women with breast cancer.

Ascorbic acid suppresses the deconjugation of noradrenaline but not dopamine in plasma.

The characteristics of hydrolysis of sulfoconjugated noradrenaline (NA) and dopamine (DA) in plasma using sulfatase were investigated. Ascorbic acid has been used as an antioxidant during the hydrolysis of conjugated NA or DA. Hydrolysis of NA sulfates was considerably inhibited by adding ascorbic acid (0.5-10 mM), and slightly inhibited by adding dithiothreitol (1-10 mM). In contrast, the hydrolysis of DA sulfates was not affected after either ascorbic acid or DTT treatment. On the basis of these findings, the levels of NA sulfates previously reported are found to be markedly lower than the actual levels of NA sulfates in human plasma.

[Activity of serum arylsulfatase B in nasal allergy patients].

Although the arylsulfatase B has been reported to inactivate slow reacting substance of anaphylaxis (SRS-A) in vitro there has not been studied about the relation between this enzyme and nasal allergy in vivo. The present study was done to examine the serum level of arylsulfatase B in 73 nasal allergy patients and 13 normal controls. Serum arylsulfatase activity was quantified by measurement of the hydrolysis product (p-nitrocatechol) generated by the interaction of this enzyme and a substrate (p-nitrocatechol sulfate, Sigma). The results are summarised as follows; 1. Arylsulfatase B activity is significantly elevated in sera of nasal allergy patients than in that of normal subjects. 2. There are no correlation between the enzyme activity and the number of peripheral blood eosinophiles. 3. There is tendency the severe the nasal obstruction, the lower the level of the enzyme activity.

[Arylsulfatase activity of asthmatic children].

Human eosinophil arylsulfatase (AS) is known to inactivate a slow reacting substance of anaphylaxis (SRS-A). Arylsulfatase A (AS-A) and arylsulfatase B (AS-B) activity was assayed by a modification of the method of Inoue using chromatography, and peripheral eosinophil cell counts were obtained to observe the circadian rhythm of 6 healthy controls and 7 children with asthma. There was no significant diurnal variation in AS between the two groups. Eosinophil counts of both groups were lower in the morning and higher at night. Theophylline and beta 2 stimulants did not affect these activities significantly. Forty asthmatic children were selected to evaluate AS activity and eosinophil counts during and after attacks. AS-B activity was significantly higher in children during attacks than at other times, 5.70 +/- 2.00 vs. 3.74 +/- 0.66 4 MUnmol/ml/2hr (p less than 0.05). This result was more evident within 24 hours of the attack (p less than 0.01). Eosinophil counts were significantly lower during attack, and there was a negative correlation between the eosinophil counts and AS-B activity. AS-B activity in mild asthmatic children was greater than in severe cases. A significant rise in AS-B was seen in EIB negative asthmatics (p less than 0.01), but no remarkable change was seen in either AS-A or AS-B in the EIB positive group. The data suggest that higher AS-B activity during asthma attacks could inactivate SRS-A and modulate allergic inflammatory reaction.

[Ichthyosis linked to the X chromosome. Biochemical and endocrinologic study]. / Ichtyose liée au chromosome X. Etude biochemique et endocrinienne.

The case of a 6-year-old male patient suffering from X-chromosome-linked ichthyosis is presented. There was no steroid sulfatase activity in the proband's leucocytes and cutaneous fibroblasts. The activity was decreased in the proband's mother's leucocytes and in one brother, affected by a mild ichthyosis. Basal plasma levels of dehydroepiandrosterone and its sulfate were normal for the patient's age, suggesting that sulfates do not play a significant role in the production of free steroids. After 3 intramuscular injections of 1,500 units of human chorionic gonadotropin, plasma levels of testosterone increased normally, indicating that there was no associated primary gonadal insufficiency.

Characterization of low-density lipoproteins from patients with recessive X-linked ichthyosis.

We investigated lipoprotein metabolism in 14 patients with recessive X-linked ichthyosis (RXLI), a metabolic disease characterized by scaly skin, corneal opacity and steroid sulfatase deficiency. Plasma total cholesterol (TC) levels ranged from normal to slightly low (mean +/- SD: 156 +/- 28 mg/dl). Four patients showed a mild or moderate elevation of plasma triglyceride (TG) levels ranging from 150 to 365 mg/dl. The apoprotein B (apo B) to TC ratio was higher than in normal controls (0.63 +/- 0.11 vs. 0.52 +/- 0.07, P less than 0.01), while plasma apoB levels were within the normal range (99 +/- 17 mg/dl). Polyacrylamide gel electrophoretic mobility of low-density lipoprotein (LDL) was markedly increased in all patients, and further analyses showed that this finding was not due to a change in the particle size of the LDL but to an increased content of cholesterol sulfate (1.0-2.3% of the LDL-cholesterol content). In addition to the alteration of electrophoretic mobility, marked changes in the lipid and apoprotein compositions of the LDL fraction were observed; cholesterol ester content in LDL (LDL-CE) was significantly lower than that of control subjects (37 +/- 4% vs. 41 +/- 2% of total lipids, P less than 0.01), while the triglyceride content (LDL-TG) and apo B to cholesterol ratios in LDL were significantly higher than those of controls (18 +/- 7 vs. 10 +/- 2, P less than 0.001; 1.21 +/- 0.19 vs. 0.73 +/- 0.05, P less than 0.001, respectively). This anionized LDL, in which cholesterol sulfate was increased, was shown to bind to the LDL receptor of fibroblasts to much the same extent as normal LDL. In conclusion, the increase in cholesterol sulfate in LDL fraction not only alters the electrophoretic moiety but also the relative contents of apoB, cholesterol, and triglyceride in the lipoprotein. It does not change the affinity of LDL for the LDL receptor.

Ichthyosis and hypogonadism in two brothers with deletion of the short arm of the X chromosome.

Two brothers were examined because of ichthyosis and hypogonadism. Their testes were small. There was no response of plasma testosterone to human chorionic gonadotropin and no response of plasma luteinizing hormone and follicle-stimulating hormone to intravenous luteinizing-hormone-releasing hormone. They both had hyposmia. Steroid sulfatase activity in white blood cells was zero. Flow cytometry and the use of special probes indicated that these two brothers had a large deletion of the short arm of the X chromosome which included the STS locus, the closely linked locus DXS237 and probably the gene for hypogonadism, findings which offer the opportunity for speculations on the locus of control of normal testicular development and function.

X-linked recessive ichthyosis. Reinvestigation of a family first described in 1928.

Recent findings in a family with X-linked recessive ichthyosis are presented. The first description of this family in the literature was given and correctly diagnosed by Csörsz in 1928. His paper can be considered one of the most widely cited proofs of the existence of X-linked ichthyosis. The extended pedigree as well as data of steroid sulfatase and arylsulfatase C determinations presented in this paper verify the diagnosis of the X-linked mode of inheritance of ichthyosis in this family. The biochemical investigations carried out on leukocytes of family members resulted not only in a confirmation of the clinico-genetic diagnosis, but they also helped to establish the heterozygous genotype of a female mentioned previously as an affected person.

Arylsulphatase C activity in leukocytes of patients and carriers of X-linked ichthyosis.

In Triton X-100 solubilized leukocytes of 17 patients and 8 obligate carriers of X-linked recessive ichthyosis (XLI) the activity of arylsulphatase C (ASC) was determined and expressed as the ratio to beta-galactosidase activity. The ASC/beta-gal ratio of XLI patients is markedly decreased (range 0.07-0.48) in comparison to the corresponding control group of males (range 1.3-2.7). The enzyme ratios of 8 obligate carriers of XLI are decreased (range 0.90-1.9) in comparison to the normal females (2.13-5.52). These results indicate that the determination of the enzyme ratio of ASC/beta-gal in Triton X-100 solubilized leukocytes is a sensitive test for biochemical identification of patients and probably of carriers of XLI.

Placental sulfatase deficiency: maternal and fetal expression of steroid sulfatase deficiency and X-linked ichthyosis.

PSD-X-linked ichthyosis are manifestations of a similar disorder of an inborn error of metabolism characterized by a deficiency of steroid sulfatase. The decreased enzyme activity is due to the absence of the expression of enzyme (steroid sulfatase) protein. Affected individuals with this disorder are males (X-linked inheritance) with a frequency of 1/2000 to 1/6000 births. Homozygous females from cosanguineous marriages have been reported with this disorder. The diagnosis is suspected and confirmed by: Low estriol excretion; Negative DHEAS loading test Increased DHEAS in amnionic fluid; Normal DHEAS in cord plasma; Possible delayed or abnormal labor patterns; Decreased sulfatase activity in the placenta, fibroblast, erythrocytes, lymphocytes or leukocytes of affected individuals; Development of ichthyosis in male infants at 2 to 3 months of age.

Hunter syndrome: prenatal diagnosis in maternal serum.

Iduronate sulfate sulfatase (ISS), the deficient hydrolase in Hunter syndrome, consistently increases in the serum of pregnant women, reaching a three- to fourfold increase from pre-pregnancy levels toward the end of pregnancy. In Hunter carriers, a correlation occurs between the status of the fetus with regard to Hunter syndrome and the ISS increase in maternal serum. Thus, in pregnancies with Hunter-affected fetuses, enzyme levels did not change in the serum of heterozygous mothers until abortion was performed, while in nonaffected fetuses, ISS increased usually very early in pregnancy--as early as the 6th-12th week. In heterozygote female fetuses, this increase might be delayed. These data imply that a prenatal diagnosis of Hunter syndrome might be accomplished in maternal serum at early conventional procedures for the prenatal diagnosis of Hunter syndrome.