Implementation of an Affordable Method for MPS Diagnosis from Urine Screening to Enzymatic Confirmation: Results of a Pilot Study in Morocco.

BACKGROUND: Rapid and accurate diagnosis of mucopolysaccharidoses (MPS) is still a challenge due to poor access to screening and diagnostic methods and to their extensive clinical heterogeneity. The aim of this work is to perform laboratory biochemical testing for confirming the diagnosis of mucopolysaccharidosis (MPS) for the first time in Morocco. METHODS: Over a period of twelve months, 88 patients suspected of having Mucopolysaccharidosis (MPS) were referred to our laboratory. Quantitative and qualitative urine glycosaminoglycan (GAG) analyses were performed, and enzyme activity was assayed on dried blood spots (DBS) using fluorogenic substrates. Enzyme activity was measured as normal, low, or undetectable. RESULTS: Of the 88 patients studied, 26 were confirmed to have MPS; 19 MPS I (Hurler syndrome; OMIM #607014/Hurler-Scheie syndrome; OMIM #607015), 2 MPS II (Hunter syndrome; OMIM #309900), 2 MPS IIIA (Sanfilippo syndrome; OMIM #252900), 1 MPS IIIB (Sanfilippo syndrome; OMIM #252920) and 2 MPS VI (Maroteaux-Lamy syndrome; OMIM #253200). Parental consanguinity was present in 80.76% of cases. Qualitative urinary glycosaminoglycan (uGAGs) assays showed abnormal profiles in 31 cases, and further quantitative urinary GAG evaluation and Thin Layer Chromatography (TLC) provided important additional information about the likely MPS diagnosis. The final diagnosis was confirmed by specific enzyme activity analysis in the DBS samples. CONCLUSIONS: The present study shows that the adoption of combined urinary substrate analysis and enzyme assays using dried blood spots can facilitate such diagnosis, offer an important tool for an appropriate supporting care, and a specific therapy, when available.

Differentiating malignant colorectal tumor patients from benign colorectal tumor patients by assaying morning urinary arylsulfatase activity.

AIM: For several decades urinary arylsulfatase (ARS) activity has been reported to be elevated in many cancers. It has been shown that urinary ARS activity may serve as a marker of tumor progression and therapy surveillance. This study was designed to evaluate the clinical application of detection of urinary ARS activity. METHODS: We used a modified precipitation method to separate ARS from urine samples. This method was easy to use and applicable for a high throughput assay. We tested and analyzed the morning urinary ARS activity in 300 normal controls, 97 patients with benign tumors and 119 with malignant colorectal tumor. RESULTS: Compared to normal male and female controls, morning urinary ARS activity was significantly higher in malignant colorectal tumor patients. Moreover, morning urinary ARS activity had a relatively high efficacy in distinguishing patients with malignant colorectal tumors from those with benign colorectal tumors. The area under the curve in the receiver operator characteristics curve analysis was 0.89 (95% CI, 0.83-0.95) and 0.87 (95% CI, 0.79-0.94) in male and female colorectal patients, respectively. CONCLUSION: These data suggest the potential application of morning urinary ARS activity to conventional clinical use.

Determination of 4-hydroxy-3-methoxyphenylethylene glycol 4-sulfate in human urine using liquid chromatography-tandem mass spectrometry.

A major metabolite of norepinephrine (NE) in brain is 4-hydroxy-3-methoxyphenylethylene glycol (MHPG). In many species, a large fraction of MHPG formed in brain is converted to the sulfate conjugate. Consequently, MHPG sulfate has been proposed as a biomarker for NE metabolism in the central nervous system. As part of the clinical trials of the monoamine oxidase inhibitor selegiline for treating cocaine addiction, we required a method for measuring urine concentrations of MHPG sulfate. Using a deuterium-labeled analogue as an internal standard, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/ MS) method for determination of MHPG sulfate in human urine. Sample preparation involves simply diluting 50 microL of urine with 1 mL of ammonium formate buffer and adding the internal standard. The sample is centrifuged, the supernate is transferred to an autosampler vial, and 10 microL is injected into the LC-MS/MS system. Standard curves from 50 to 10,000 ng/mL are generated. Only one sample of 277 clinical samples analyzed had a concentration outside of this range. Precision (coefficient of variation) ranged from 1.9 to 9.7%, and accuracy ranged from 97 to 103% of expected values for controls prepared by spiking sulfatase-treated urine with MHPG sulfate.

[Changes in arylsulphatase activity (EC 3.1.6.1) in the blood serum and urine in women during pregnancy and in the course of delivery]. / Zmiany aktywnosci arylosulfatazy A (EC 3.1.6.1) w surowicy i moczu kobiet w przebiegu ciazy i porodu fizjologicznego.

By the determination of arylsulphatase A activity (EC 3.1.6.1) in the blood serum and urine obtained from 66 women using the modified method by Lee-Vaupel and Conzelmann it was noticed the increase in the enzyme activity during the pregnancy comparing to the non-pregnant group. The highest enzyme activity was observed in the III trimester of pregnancy. In the following stages of delivery (I, II, III) it was assumed the increase in enzyme activity in urine. The highest enzyme activity in urine was observed in the stage III, and in the serum--in the stage II. It was compared the enzyme activity in primiparae and multiparae proving, that in the serum nd urine this activity is higher in the stages I and II in multiparae, and in the stage III in primiparae.

[Morphological and biochemical studies of the effect of the peat-derived preparation PF-290/II/2 on the development of natural lymphatic leukemia in mice]. / Badania morfologiczne i biochemiczne nad wplywem preparatu torfopochodnego PF-290/II/2 na rozwój naturalnej bialaczki limfatycznej u myszy.

AKR mice highly susceptible to leukemia were fed orally for 9 months every days with a water solution of peat-liking preparation PF-290/II/2 at a dose 0.2 cm3 (70 g/cm3 water). After bleeding body and internal organs weight were measured and their ratio were calculated. Anatomo-pathological lesions, histopathological and ultrastructural examinations with the use of transmission and scanning microscope, serum cobalt-activated acylase (AA-Co) activity and urine arylsulphatase (ASA) activity were performed. It was found used preparation had some anti-tumors effect of mice with lymphatic leukemia. Serum cobalt-activated acylase and urine arylsulphatase of AKR mice for observation on disease development and dynamics of this process. In the ultrastructural picture changes of lymphatic cells after outside removal of degradated complexes of intracell membranes was observed.

Determination of p-nitrophenol in serum and urine by enzymatic and non-enzymatic conjugate hydrolysis and HPLC. Application after parathion intoxication.

In connection with the toxicologic analysis of a number of parathion intoxications a method for determination of free and conjugated forms of p-nitrophenol (p-NP) as the main metabolite of parathion in blood and urine was established. Quantification of conjugates is based on their hydrolysis followed by detection of p-NP using a sensitive HPLC method. Hydrolysis of both p-NP-glucuronide and p-NP-sulfate is performed by specific enzymes and also by mineral acid, the latter is also found to be highly selective under definite conditions. The two hydrolysis methods applied showed a good correlation. The levels of free and conjugated p-NP in series of blood and urine samples were established after survival from two parathion intoxications. The individual levels of p-NP-sulfate and p-NP-glucuronide in both cases are discussed in respect of results made by other authors in this field.

[Determination of enzyme activity and beta 2-microglobulin concentration in the urine in chronic diffuse glomerulonephritis and hypertension]. / Opredelenie aktivnosti fermentov i kontsentratsii beta 2-mikroglobulina v moche bol'nykh khronicheskim diffuznym glomerulonefritom i gipertonicheskoi bolezn'iu.

Activity of alanine aminopeptidase (AAP), aryl sulphatase A (ASA) beta-glucoronidase (beta-GA) and the concentration of beta 2-microglobulin in urine was determined in 12 patients with chronic diffuse glomerulonephritis and in 16 patients with essential hypertension. The control group consisted of 6 practically healthy persons. The AAP activity in urine of patients with chronic glomerulonephritis was significantly higher than that in healthy subjects. Renal excretion of lysosomal enzymes (ASA, beta-GA) appeared to be less expressed; no significant differences as regard to their excretion were noted between the above mentioned groups. Concentrations of beta 2-microglobulin in urine of patients with chronic glomerulonephritis 6 times exceeded its concentration in healthy persons and in hypertensive patients, more than 3 times as much. Determination of the enzyme activity and beta 2-microglobulin concentration in urine may serve a test in differential diagnosis, for evaluation of the treatment efficiency of patients with chronic glomerulonephritis accompanied by arterial hypertension and those suffering from essential hypertension.

[Metabolic aspects of parsalmide in the cat (author's transl)]. / Untersuchungen uber den Stoffwechsel von Parsalmid bei der Katze.

2-Proparglyoxy-5-amino-N-(n-butyl)-benzamide(parsalmide, My 41-6) given s.c. to the cat and excreted in the urine, was present partly as unchanged parsalmide (25-62% of total excreted compounds), partly as unstably conjugated compounds (17-36%, mainly N-glucuronilparsalmide), and partly as stably conjugated compounds (11-36%, mainly N-acetylparsalmide). Urinary excretion of parsalmide and its metabolites continued for some days, indicating a slow excretion from the body, contrasting with other species, man included, previously examined. Summarizing, there are some 5-6 metabolites of parsalmide in the urine of treated cats, apart from unchanged parsalmide.

Adult metachromatic leukodystrophy. III. Clinical course, final stages and first biochemical results.

Continuing the previously published clinical development of a case of adult metachromatic leukodystrophy (MLD), we now describe the terminal phase and death (at 46 years of age) of our patient. The final phase was characterized clinically by progression of generalized peripheral neuropathy, advanced extrapyramidal and pyramidal tract symptomatology, dementia and brainstem dysfunction. First biochemical results show a moderate relative increase (3- to 5-fold) of sulfatides in the frontal lobe white matter but not in the cortex. The analysis of fatty acids in total lipid extract shows a decrease of long-chained fatty acids in favor of short-chained fatty acids, this change is more pronounced in white matter in the cortex. The clinical course and biochemical results are discussed in relation to previous cases analyzed by us. Epidemiological aspects especially emphasize routine serach for MLD amongst patients with neuropsychiatric symptomatology showing unusual psychoses, presenile dementias or unspecific disturbance of motor coordination possibly with electroneurographic evidence of peripheral neuropathy.

Urinary excretion of arylsulfatases in malnourished/vitamin A deficient children.

Serum vitamin A (retinol) levels were generally low in all malnourished children (6-15 microgram/100 ml) compared with control children (50 microgram/100 ml). A significant increase in vitamin A after appropriate therapy was observed in all malnourished groups. Dietary supplements of proteins and calories even without extra vitamin A supplements increased serum vitamin A levels in cases of kwashiorkor indicating active mobilization of liver vitamin A. Total urinary arylsulfatase A activity excreted in 24-h or within 8-h in the morning (6 a.m. to 2 p.m.) was significantly reduced in cases of malnutrition with or without mild vitamin A deficiency symptoms. The excretion of arylsulfatase B was not altered. In cases of severe vitamin A deficiency coupled with malnutrition increased excretion of both arylsulfatases A and B was evident. These results on urinary arylsulfatases excretory pattern have been obtained either in samples collected for 24-h or specifically for 8-h (morning) and it is suggested that this test on urinary arylsulfatases may prove useful for detection of acute vitamin A deficiency with malnutrition in field studies. A ratio of arylsulfatases A/B of 2.0 or less seems to indicate mild malnutrition, the normal ratio being 3.4. Furthermore a low ratio coupled with increased excretion of both arylsulfatases A and B may be considered specific for acute vitamin A deficiency.

Urinary acidic hydrolases in renal diseases in children.

Acidic hydrolases were assayed in urines of 19 normal children, 33 children with idiopathic nephrotic syndrome of childhood (INS), 21 children with glomerulonephritides (GN) and 7 children with persistent proteinuria/hematuria, and in plasma of 10 children each with INS or GN. Both plasma and urinary acidic hydrolases were studied in intermittent orthostatic proteinuria. Cbeta-galactosidase and Cbeta-N-hexosaminidase were done in normals and children with active renal disease. Significantly (P less than 0.01) elevated urinary acidic hydrolases excretion in active renal diseases, both in INS and GN, returned to a normal range with regression of the diseases. Increased postural proteinuria was associated with normal urinary acidic hydrolases. Both beta-galactosidase and beta-N-hexosaminidase excretion was higher than similar mol wt proteins in normals and increased further in active renal diseases. The data suggests that increased urinary acidic hydrolases is related to the activity of the renal disease, and not to urinary WBC, hematuria or proteinuria. The likely source of urinary acidic hydrolases thus appears to be the injured renal parenchyma itself.

Methyl-CCNU versus methyl-CCNU and 5-fluorouracil in carcinoma of the large bowel.

5-Flourouracil (5-FU) and methyl-CCNU have demonstrated separate sensitivities in carcinoma of the large bowel. This study was an attempt to see if methyl-CCNU versus methyl-CCNU plus 5-FU would demonstrate different responses in advanced colorectal carcinoma. Forty-nine patients have been evaluated, 14 receiving methyl-CCNU and 35 receiving 5-FU plus methyl-CCNU. One partial response has been seen with methyl-CCNU alone in a patient with liver metastasis. Thirteen partial responses have been noted in patients treated with the two-drug combination. There was a significant difference in the median survival of the responders versus the nonresponders for the two-drug group. Side effects were expected: nausea and vomiting, leukopenia, and thrombocytopenia. Plasma carcinoembryonic antigen and urine arylsulfatase were measured in all patients and correlated well with response.

Arylsulfatases in bilharziasis.

Arylsulfatase A and B in urine have been estimated in 18 normal subjects and 50 bilharziasis patients. The bilharziasis patients were divided into two groups according to the type of infeciton. Those with bilharziasis haematobian type of infection and those with the bilharziasis mansoni type. Each group was further subdivided into subgroups according to the severity and progress of the disease. The activities of arylsulfatase A and B were significantly elevated in all the groups of patients studied and it is evident that there is a progressive increase with the progress of the disease in both types of bilharziasis infections (the haematobian and mansoni types). Liver dysfunction consequent of bilharzial infestation appears to take part in the mechanism of induction of the bilharzial bladder cancer.

[The effect of therapeutic gentamycin doses on the enzyme secretion in urine]. / Der Einfluss therapeutischer Gentamyzindosen auf die Enzymausscheidung im Harn

8 patients with chronic pyelonephritis were given gentamycin intramuscularly injected in individual dosage during 8-10 days. Here the behaviour of the excretion of protein, alanine aminopeptidase alkaline phosphatase, alpha-glucosidase, gamma-glutamyl transpeptidase and lysozyme with the urine was tested. With the exception of the lysozymuria, which increased only in patients with chronic renal insufficiency, regularly a hyperenzymuria developed. Most distinctly the excretion of the alanine aminopeptidase increased. After initial decrease the excretion of total protein transiently increased after completion of the gentamycin therapy. All the deviations were reversible. From the increased excretion of enzymes may not be concluded to a nephrotoxicity of gentamycin.

[Enzymes in urine following administration of hypertonic solutions]. / Enzyme im Harn nach Applikation hypertoner Lösungen

3 patients with chronic nephropathies were given 20 ml of a diatrizoate-X-ray contrast medium, 500 ml of a 10% mannitol solution and 500 ml of a 10% dextran solution intravenously, and the behaviour of the excretion of protein, alanine aminopeptidase, beta-glucuronidase, aryl sulphatase A and lysozyme with the urine was tested. After application of these substances a transient increase of the excretion of alanine aminopeptidase, aryl sulphatase A and protein takes place. Conspicuous is the temporary decrease of the beta-glucuronidase activity in the urine after application of these hypertonic solutions. As a common cause of these changes alterations of the tubular cell in the sense of an osmotic nephropathy are to be assumed.