Patterns of a Rectal Microbicide Placebo Gel Use in a Preparatory Stage for a Phase I Trial Among Young Men Who Have Sex with Men.

We examined young gay, bisexual, and other men who have sex with men's (YGBMSM) usage patterns of a pre-coital, applicator-administered rectal placebo gel. An ethnically diverse sample of 94 YGBMSM (aged 18-30 years) were asked to insert hydroxyethylcellulose placebo gel rectally before receptive anal intercourse (RAI) and report their gel use through an interactive voice response system (IVRS) across 12 weeks. We used trajectory analyses to characterize participants' use of the rectal gel over the 12 weeks, and examine whether these trajectories varied based on participants' sociodemographic characteristics, sexual behaviors, application and insertion behaviors, and experiences using the placebo gel. A cubic model was the best fit for these longitudinal data, with two distinct trajectories of gel use observed. The first trajectory ('High with Varying Gel Use per Week') represented YGBMSM (N = 38; 40.3%) who reported using the rectal gel on several occasions per week. The second trajectory ('Low and Consistent Gel Use per Week') represented participants (N = 56; 59.7%) who reported a consistent average use of one gel per week. Participants in the High with Varying Gel Use Trajectory reported trying out a greater number of positions when inserting the gel across the 12-weeks than peers in the Low and Consistent Gel Use Trajectory. YGBMSM reporting more RAI occasions during the trial were more likely be present in the High with Varying Gel Use Trajectory than peers in the Low and Consistent Gel Use Trajectory. Future research examining how to facilitate gel application and adherence among YGBMSM is merited.

To Use a Rectal Microbicide, First Insert the Applicator: Gel and Applicator Satisfaction Among Young Men Who Have Sex With Men.

We examined how experiences with a rectal placebo gel and applicator used with receptive anal intercourse (RAI) related to young men who have sex with men's (YMSM) likelihood of using a rectal microbicide gel and applicator in the future. An ethnically diverse sample of 95 YMSM (aged 18 to 30 years) were asked to insert hydroxyethylcellulose (HEC) placebo gel rectally before RAI during 12 weeks and report the product's acceptability (i.e., satisfaction with applicator and gel, respectively; perceived gel side effects; and sexual satisfaction when gel was used) and likelihood of future microbicide use. Main and interaction effects predicting future use intentions were tested using linear regression. We found a positive association between future use intentions and applicator satisfaction (b = .33, p < .001). In a subsequent interaction effects model, we found that greater gel satisfaction was associated with increased future use intentions; however, the strength of this relationship was magnified when YMSM reported greatest satisfaction with the rectal applicator. Applicator satisfaction may be a salient factor in YMSM's decision-making to use a rectal microbicide in the future. Although the importance of developing a satisfactory rectal microbicide gel for YMSM is undeniable for its future use, our results also emphasize the importance of developing strategies that increase YMSM's comfort and skill when using a rectal applicator. Future research examining how to optimize the design, properties, and characteristics of a rectal applicator as a strategy to promote greater satisfaction and use among YMSM is merited.

Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme alpha-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb(3)), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb(3) in urine samples collected on filter paper, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb(3) techniques being both time- and labour-intensive, this filter paper method eliminates lipid extraction, glycolipid isolation, centrifugation and evaporation steps, while maintaining sensitivity and efficiency. The stability of Gb(3) on filter paper was good for a 7-week period under different temperature conditions. Normal control values were established and the technique was tested with anonymized samples from Fabry hemizygotes and heterozygotes. The levels of total Gb(3) in all classical hemizygotes were well above the control values and all heterozygotes, except two nonexcretors, were above the reference level. The proposed novel filter paper method favours the collection, storage and shipment of samples. It is simple and efficient for a feasibility study, potentially applicable to the determination of total urinary Gb(3) in the newborn population as part of a screening programme, and could also be used in high-risk screening laboratories. Since the incidence of Fabry disease is hard to establish, owing to the heterogeneous clinical expression of the visible phenotype, this feasibility study could help determine its actual incidence in the Quebec population.

Newborn urine screening programme in the province of Quebec: an update of 30 years' experience.

The introduction of our voluntary mass screening programme in 1971, in the province of Quebec, has permitted us to detect different inborn errors of metabolism in the newborn population using a thin-layer chromatographic (TLC) technique with sequential use of different sprays on the same plate. Abnormalities in amino acids and organic acids are detected in urine filter paper specimens of 21-day-old babies. Initial parental compliance is 90% and climbs to 99.25% for repeat sample requests. Screening is centralized in one laboratory, while diagnosis, counselling, management and follow-up are done in four regional centres. Over 25 inherited Mendelian disorders can be identified. There have been certain modifications in our programme throughout the years in order to increase efficiency, screen for a larger number of disorders, improve the quality of the collection of the urine filter paper samples, increase parental compliance and better manage the data bank. However, one goal has remained a priority: early prevention of genetic diseases. We present an overall view of our screening programme with an add-on technique to detect different organic acidurias, our recent statistics and the modifications implemented over the years.

Outcome of individuals with low-moderate methylmalonic aciduria detected through a neonatal screening program.

BACKGROUND: The clinical spectrum of methylmalonic aciduria (MMAuria) ranges from severe, neonatal acidosis to benign asymptomatic organic aciduria. In 1975, screening for MMAuria was established in the province of Quebec. Although newborn screening programs facilitate presymptomatic detection and treatment and also detect asymptomatic variants, uncertainties about potential long-term hazards of mild to moderate elevations of MMA create concern. The objective of this study was to examine the outcome of individuals excreting low to intermediate quantities of MMA, ascertained by a newborn screening program. RESULTS AND STUDY DESIGN: One hundred and thirty-six individuals with elevations of urinary MMA were initially identified by the screening program; 122 individuals were noted to have excretion of urinary MMA <1400 micromol/mmol creatinine. At follow-up assessment at 1 year of age, in 65 of these 122 individuals, the MMA excretion had resolved. Of the remaining individuals, 9 were lost to follow-up, 13 had symptoms, and the remaining 35 were free of symptoms. Among the 35 individuals with asymptomatic persistent MMAuria, MMA excretion has resolved in 13 over 1 year; 22 individuals exhibit persistent low-moderate MMAuria (range, 210 to 1133 micromol/mmol creatinine). CONCLUSION: Follow-up examination of individuals in the latter asymptomatic cohort with persistent low-moderate MMAuria indicates normal somatic and cognitive outcomes.

Stability of urinary HVA and VMA on filter paper.

The study examined the stability of HVA and VMA in 1-ml aliquots of a single urine sample stored on filter paper at different temperatures for 2 years. The results showed that HVA and VMA were stable in dried filter paper when stored at 4 degrees C or lower temperature. Storage at room temperature resulted in degradation of the sample.

Methylmalonic acid quantification by stable isotope dilution gas chromatography-mass spectrometry from filter paper urine samples.

A specific method for quantification of methylmalonic acid (MMA) from urine samples dried onto filter paper is described. The method involves stable isotope dilution gas chromatography-mass spectrometry with [methyl-2H3]-MMA as the internal standard. MMA is stable in dry paper samples stored at room temperature for at least 2 weeks. The extraction efficiency of MMA from paper was 56-58%. The concentration of urinary MMA in dried filter paper specimens from 190 normal controls was 1.21 +/- 1.34 (mean +/- SD) mmol/mol of urinary creatinine. Age-related reference values are also reported. The concentrations, normalized to the urinary creatinine concentration, decrease with age. The applicability of this method to rapid screening for cobalamin (vitamin B12)-related disorders and methylmalonic aciduria is demonstrated.

Prenatal diagnosis of Smith-Lemli-Opitz syndrome is possible by measurement of 7-dehydrocholesterol in amniotic fluid.

Amniocentesis was performed at 17.3 weeks in a pregnancy with severe intrauterine growth retardation. Cytogenetic studies on amniocytes were normal, 46,XX, and the pregnancy was continued. The diagnosis of Smith-Lemli-Opitz syndrome was suspected in the neonatal period and confirmed by the presence of 7-dehydrocholesterol (7-DHC) in the plasma (0.4 mmol/l, normal = not detectable) associated with a low total cholesterol concentration (0.4 mmol/l, normal = 2.56 +/- 0.23). Retrospective analysis of the amniotic fluid sample revealed an elevated level of 7-DHC (0.022 mmol/l; normal = undetectable). Therefore measurement of 7-DHC levels in amniotic fluid during the second trimester of pregnancy is useful for the prenatal diagnosis of Smith-Lemli-Opitz syndrome in families at risk and should be considered in cases of severe growth retardation of unknown aetiology for which amniotic fluid is available and in which a normal chromosomal pattern in amniocytes is present.

A profile of cerebral and hepatic carnitine, ammonia, and energy metabolism in a model of organic aciduria: BALB/cByJ mouse with short-chain acyl-CoA dehydrogenase deficiency.

Spontaneous animal models of inborn errors of metabolism are valuable tools for defining the pathogenesis of these disorders and also the mechanism of various therapeutic approaches. In the present study, we have employed BALB/cByJ mice with an autosomal recessive deficiency of short-chain acyl-CoA dehydrogenase (SCAD). These animals were characterized by a marked urinary excretion of ethylmalonic and methylsuccinic acids along with butyrylglycine. Using adult homozygous mice we have studied the basic cerebral and hepatic profile of carnitine, ammonia, and energy metabolism. The effects of fasting and a short-term supplement of L-carnitine have been evaluated in comparison with control BALB/cJ mice. The mutant mice had low levels of acetyl-CoA and high levels of lactate compared to control mice. Fasting aggravated this condition by further decreasing acetyl-CoA and increasing lactate levels in the mutant mice. Free carnitine levels were significantly decreased in liver with fasting. Long-chain acylcarnitines were significantly lower in the brain of mutant mice. A short-term supplementation of L-carnitine resulted in general increases of carnitine levels in liver and muscle, but they still remained lower in mutant BALB/cByJ mice as compared to control BALB/cJ mice. L-Carnitine treatment increased cerebral CoA-SH levels and both hepatic and cerebral acetyl-CoA levels in mutant mice. Hyperammonemia which has been described frequently in acyl-CoA dehydrogenase deficiencies was not observed in adult BALB/cByJ mice. This could be due to a rapid conjugation of butyryl-CoA with glycine by an increased activity of glycine N-acyltransferase.

Breeding experiments to combine the X-linked sparse-fur (spf) mutation with the autosomal recessive BALB/cByJ strain: testing the biochemical phenotype of double-mutant mice as a model for ammonia: fatty acyl CoA synergism.

Breeding experiments were conducted to combine the X-linked sparse-fur (spf) mutation with ornithine transcarbamylase deficiency and the autosomal recessive deficiency of short-chain acyl CoA dehydrogenase (SCAD) in BALB/cByJ mice. We obtained spf/Y (scad/scad), spf/+ (scad/scad) and spf/spf (scad/scad) double mutants amongst the F2 progeny, which were tested and separated on the basis of urinary orotate and the GC/MS analysis of urinary butyrylglycine, methylsuccinate and ethylmalonate. The testing of the biochemical type was feasible both on the basis of a 24-h urine collection form adult mice kept in metabolic cages and on the basis of urine spots collected on filter paper from younger progeny. It is postulated that the spf/Y (scad/scad) double-mutant may serve as a useful animal model to study the ammonia: fatty acyl CoA synergism.

Screening urine of 3-week-old newborns: transient methylmalonic and hydroxyphenyllactic aciduria.

Urinary methylmalonic acid (MMA) and 4-hydroxyphenyllactic acid (HPL) have been determined in 3345 and 2498 3-week-old newborns, respectively. Urine was collected onto filter paper and assayed by a rapid gas chromatography-mass spectrometry method. Forty-six infants (1.7%) had elevated MMA levels (greater than 58.5 micrograms/mg creatinine, means + 5 SD) and 31 infants (1.2%) had elevated levels of HPL (greater than 87.7 micrograms/mg creatinine, means + 5 SD). Fifteen infants with elevated values of MMA were retested from one to several months after the first test. In 12 infants the MMA levels normalized, while in the remaining three, elevated methylmalonic acid persisted. Nine infants with elevated values of HPL were retested, and in all except one, HPL levels normalized. No access to clinical evaluation of the infants was available. Transient methylmalonic aciduria and transient tyrosyluria affect a substantial number of infants and the clinical significance of this phenomenon has yet to be determined.

Influence of debrisoquine phenotype and of quinidine on mexiletine disposition in man.

Mexiletine is a low clearance drug which undergoes extensive metabolism in man. In vitro studies with human liver microsomes have suggested that major oxidation pathways of mexiletine are predominantly catalyzed by the genetically determined debrisoquine 4-hydroxylase (cytochrome P450IID6) activity. In this study, we investigated the role of debrisoquine polymorphism and the effects of low dose quinidine, a selective inhibitor of cytochrome P450IID6, on the disposition of mexiletine. Fourteen healthy volunteers, 10 with the extensive metabolizer (EM) and 4 with the poor metabolizer (PM) phenotype, received a single 200-mg dose of mexiletine hydrochloride orally on two occasions (1 week apart), once alone and once under steady-state conditions for quinidine (50 mg QID). During the phase mexiletine alone, total clearance, nonrenal clearance and partial metabolic clearance of mexiletine to hydroxymethylmexiletine, to m-hydroxymexiletine and to p-hydroxymexiletine were decreased in PM compared to EM (all P less than .05). In EM, quinidine decreased mexiletine total clearance from 621 +/- 298 to 471 +/- 214 ml/min (mean +/- S.D.; P less than .05) and mexiletine nonrenal clearance from 583 +/- 292 to 404 +/- 188 ml/min (P less than .05). Moreover, quinidine increased mexiletine elimination half-life in EM from 9 +/- 1 to 11 +/- 2 h (P less than .05). In these subjects, partial metabolic clearance to hydroxymethylmexiletine, m-hydroxymexiletine and p-hydroxymexiletine were decreased by quinidine coadministration 5-, 4- and 7-fold, respectively, whereas partial metabolic clearance to N-hydroxymexiletine was unaffected. Changes induced by quinidine in EM were correlated to their debrisoquine metabolic ratio. Thus, genetically determined or pharmacologically induced modulation of cytochrome P450IID6 activity represents a major determinant of mexiletine disposition.

The effect of taurine on the cholestatic potential of sulfated lithocholate and its conjugates.

The present study was aimed at determining whether the protection by taurine of lithocholate-sulfate-induced cholestasis is mediated by conjugation or by direct effect of the amino acid on bile formation. Injection of free and conjugated (glycine and taurine) sulfated lithocholate in guinea pigs significantly reduced the secretion rate of non-sulfated bile acids in bile. There was no decrease in bile flow after the injection of taurine-conjugated sulfated lithocholate, which was completely recovered in bile within 60 min. In contrast, injection of sulfated lithocholate and its glycine conjugate led to a marked decrease in bile flow, and neither one was significantly recovered in bile. In addition, both caused morphological changes in the liver, characterized by the accumulation of cytoplasmic vacuoles with lamellated myelin figures characteristic of phospholipidosis. Pretreatment with taurine (0.5% in drinking water for 3 days) prevented both the drop in bile flow and the histological changes in the liver, suggesting that conjugation with taurine removed the cholestatic potential of sulfated lithocholate. However, since taurine was effective not only in preventing cholestasis induced by the free form of sulfated lithocholate but also against its glycine conjugate, these results suggest that other mechanisms in addition to conjugate must be involved.

Effect of taurine on total parenteral nutrition-associated cholestasis.

A decrease in the formation/secretion of bile has been well documented in animals on total parenteral nutrition (TPN). Either an excess or an imbalance of amino acids (AA) has been most often implicated. In view of recent work showing that taurine promotes bile flow, bile acid secretion, and protects against hepatotoxic bile acids, the effect of adding taurine (15 mg/dL) to an AA solution was examined in guinea pigs on TPN for 3 days. The TPN-taurine group had a larger bile flow than the group without taurine and had bile acid secretory rates (BASR) similar to those of controls who were on saline by central catheter and had free access to food. Bile composition showed an increase in the secondary bile acid, 7-ketolithocholate and a concomitant decrease in chenodeoxycholate (CDC) in both experimental groups. Taurine led to a reversal of the usual predominance of glycine over taurine conjugated bile acids as well as to increases in HCO3 in cholesterol secretion. In response to a challenge with a large load of CDC, the TPN-taurine animals increased their BASR beyond those observed in the two other groups. These observations suggest that the addition of taurine to TPN solutions could play a role in the prevention of altered biliary function associated with AA solutions.

Screening for neuroblastoma at 3 weeks of age: methods and preliminary results from the Quebec Neuroblastoma Screening Project.

A large neuroblastoma screening study was recently started in the province of Quebec, Canada. This project, a collaboration between the Quebec Network for Genetic Medicine and the University of Minnesota, is studying the impact of screening infants for the preclinical detection of neuroblastoma on the population-based mortality caused by this tumor. All infants born in Quebec during a 5-year period will be screened twice, at 3 weeks and at 6 months. Urinary homovanillic acid and vanillylmandelic acid determination from dried filter paper samples is used for screening. Initial qualitative screening is done by means of thin-layer chromatography with confirmatory quantitative screening by gas chromatography-mass spectrometry (GC-MS). During the initial 6 months of 3-week screening, 41,673 neonates (92% compliance rate) were screened and 10.6% of them were tested also by GC-MS. Nine of these neonates had positive results on two GC-MS tests and were referred for evaluation to rule out the presence of neuroblastoma. Four had the tumor, 1 had a calcified adrenal gland, and 4 had no tumor detected. Three additional neonates had clinical diagnosis of neuroblastoma before they reached the screening age of 3 weeks. A neuroblastoma that did not secrete homovanillic acid or vanillylmandelic acid was diagnosed clinically in 1 additional patient who tested negative by screening.

A IIIman protein is involved in the transport of glucose, mannose and fructose by oral streptococci.

We show in this article that the transport of glucose, mannose and fructose by the phosphoenolpyruvate: mannose phosphotransferase system of oral streptococci requires the participation of a protein component that we have called IIIman. This protein was purified from Streptococcus salivarius by chromatography on DEAE-cellulose, DEAE-TSK, hydroxyapatite, and Dyematrex Green A. The purified protein migrated as a 38,900 molecular weight protein on a sodium dodecyl sulfate polyacrylamide gel. However, electrophoretic analysis of phosphoproteins and Western blot experiments indicated the presence in membrane-free cellular extracts of S. salivarius of 2 different forms of IIIman having molecular weights of 38,900 and 35,200. The presence of the high-molecular-weight form of IIIman was observed by immunodiffusion, Western blot and phosphorylation by [32]PEP in S. salivarius, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus lactis but not in Streptococcus faecium, Staphylococcus aureus, Bacillus subtilis and Lactobacillus casei. Antibodies directed against the IIIman of S. salivarius did not react with the IIIman of Escherichia coli.