Maternal screening for hypothyroidism and thyroiditis using filter paper specimens.

BACKGROUND AND OBJECTIVE: Hypothyroidism and autoimmune thyroiditis are more prevalent than previously considered in women during pregnancy and the postpartum, and are associated with adverse effects on the mother and her fetus. We determined the efficacy and accuracy of screening women for primary hypothyroidism and autoimmune thyroiditis by testing TSH and two thyroid antibodies (TAb): thyroperoxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb), in eluates of filter paper specimens collected during early pregnancy and the postpartum. METHODS: We enrolled 494 first-trimester pregnant women with no exclusion criteria into a prospective study to detect primary hypothyroidism and autoimmune thyroiditis. Finger stick blood was applied to filter paper, dried in room air, eluted, and promptly tested for TSH and TAb. A total of 178 of the pregnant women (36%) were tested in the early postpartum. Women with abnormal results had confirmatory serum tests. RESULTS: It was found that 91 pregnant women (18.4%) and 43 postpartum women (24.2%) had abnormal TSH values (>4.0 mU/L) and/or positive TAb; 140 pregnant women (28.3%) had TSH values >2.5 mU/L. All subjects with TSH values >4.0 mU/L tested positive for TAb. Eighteen women (3.6%) who tested normal during pregnancy tested abnormal in the postpartum. CONCLUSIONS: This study confirms that TSH and TPOAb measured in eluates of blood-spotted filter paper specimens are excellent screening tests to detect primary hypothyroidism and autoimmune thyroiditis in pregnant and postpartum women. Results are very comparable to serum data in this population published in the literature.

A limited spectrum of phenylalanine hydroxylase mutations is observed in phenylketonuria patients in western Poland and implications for treatment with 6R tetrahydrobiopterin.

Phenylketonuria (PKU) is an autosomal recessive defect in hepatic metabolism of phenylalanine, which is secondary to mutations in the phenylalanine hydroxylase (PAH) gene. Sixty-seven ethnically Polish PKU patients, followed at the Outpatient Department of Pediatrics and Developmental Medicine in Poznan, Poland, were assessed for mutations in the PAH gene. Two mutations were identified in 61 of 67 patients and a single mutation was identified in the remaining six patients. The four most prevalent mutations (p.R408W, 68%; c.1066-11G>A, 6%; c.1315+1G>A, 5.2%; c.822-832delGCCCATGTATA, 3.7%) accounted for 83% of the mutant alleles. Fifteen additional mutations were identified of which most (13/15) were observed in an individual patient. Before knowledge of PAH genotypes, 19 patients were challenged with a 20 mg kg(-1) dose of 6R tetrahydrobiopterin (BH(4)) and serum phenylalanine concentration was monitored in hospital over 24 h. Two patients responded to the BH(4) challenge with a reduction of serum phenylalanine concentration >30% from baseline. PAH genotypes of the two responsive patients would have been predicted, as they contained mutations recognized as BH(4) responsive, whereas the 17 patients who were unresponsive would have been predicted as their mutations were either recognized as non-responsive or were highly deleterious frame-shift mutations. Overall, only 7.5% (5/ 67) of patients had PAH mutations recognized as responsive to co-factor therapy. Among the PKU patients from western Poland, PAH mutations responsive to BH(4) therapy are poorly represented; therefore, genotyping may be useful for identifying candidate patients likely to respond to BH(4) before physiological challenge.

Biochemical characterization of mutant phenylalanine hydroxylase enzymes and correlation with clinical presentation in hyperphenylalaninaemic patients.

The biochemical properties of mutant phenylalanine hydroxylase (PAH) enzymes and clinical characteristics of hyperphenylalaninaemic patients who bear these mutant enzymes were investigated. Biochemical characterization of mutant PAH enzymes p.D143G, p.R155H, p.L348V, p.R408W and p.P416Q included determination of specific activity, substrate activation, V(max), K(m) for (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), K (d) for BH(4), and protein stabilization by BH(4). Clinical data from 22 patients either homozygous, functionally hemizygous, or compound heterozygous for the mutant enzymes of interest were correlated with biochemical parameters of the mutant enzymes. The p.L348V and p.P416Q enzymes retain significant catalytic activity yet were observed in classic and moderate PKU patients. Biochemical studies demonstrated that BH(4) rectified the stability defects in p.L348V and p.P416Q; additionally, patients with these variants responded to BH(4) therapy. The p.R155H mutant displayed low PAH activity and decreased apparent affinity for L-Phe yet was observed in mild hyperphenylalaninaemia. The p.R155H mutant does not display kinetic instability, as it is stabilized by BH(4) similarly to wild-type PAH; thus the residual activity is available under physiological conditions. The p.R408W enzyme is dysfunctional in nearly all biochemical parameters, as evidenced by disease severity in homozygous and hemizygous patients. Biochemical assessment of mutant PAH proteins, especially parameters involving interaction with BH(4) that impact protein folding, appear useful in clinical correlation. As additional patients and mutant proteins are assessed, the utility of this approach will become apparent.

Assays for thyroid-stimulating hormone using dried blood spotted filter paper specimens to screen for hypothyroidism in older children and adults.

OBJECTIVE: Symptoms of hypothyroidism in adults can be mistaken for medical and psychiatric diseases, as well as for general signs of ageing such as weakness, lethargy and fatigue. The incidence of hypothyroidism is many-fold higher in adults than in newborn children. The latter have been routinely screened for the condition using filter paper dried blood spots (DBS) for nearly three decades but this cost-effective screening technique has only recently been applied to adults. This study was undertaken to show that DBS testing in adults and older children is an accurate way to screen for hypothyroidism. METHODS: Serum and DBS specimens were collected from adults and children. Assays were run on both specimens and the results correlated. In addition 972 specimens were collected from adults at community centres and nursing homes. Follow-up studies were performed on patients with positive results. RESULTS: The correlation coefficient for 118 matched serum and DBS specimens was 0.99. Thyroid-stimulating hormone (TSH) values were elevated in 50 of the 972 adults from nursing homes and community centres. Thirteen of these individuals were on thyroid medication and 28 had either high serum TSH or high thyroglobulin (TgAb) or thyroid peroxidase (TPOAb) antibody levels. CONCLUSIONS: Individuals can be screened for hypothyroidism by collecting finger stick DBS specimens at community centres, nursing homes and other locations which can be mailed by regular postal service to a central laboratory for accurate and inexpensive testing.

How do people with Type 2 diabetes perceive and respond to cardiovascular risk?

OBJECTIVES: To explore how people with Type 2 diabetes perceive cardiovascular risk, and how those perceptions might affect their motivation to make lifestyle changes. METHODS: The setting was a diabetes clinic in a UK teaching hospital. A qualitative study was conducted, using semistructured individual interviews and template analysis of content. The participants were 20 Type 2 diabetic patients, aged between 52 and 77 years, half with and half without cardiovascular disease (CVD). RESULTS: Whether they had CVD or not, most people were aware they were at risk of it, of its causative factors, and possible effects. However, they were more likely to attribute it to unchangeable factors like 'stress' and 'heredity', than medical risk factors like cholesterol and smoking. Patients with pre-existing CVD correctly regarded their risk as higher than those without. Few saw any direct link between being diabetic per se and cardiovascular risk. Lifestyle changes were precipitated by major life events and motivated by family support, fear of complications, and a belief that one should follow doctors' advice. A common reaction to CVD and diabetes was stoical acceptance, allowing patients to view their lives positively, whilst living with unpredictable potentially disabling diseases. CONCLUSIONS: Patients were unaware how strongly diabetes influences cardiovascular risk. Their ideas about risk were very different from those of conventional medicine, and provided individual rationales for making choices about treatment and risk-influencing behaviour. Contextual factors, such as family milieu, also influenced their behaviour. Clinicians should not assume patients share the same mental model of risk as they, and must be prepared to explore peoples' individual constructs and health beliefs.

Superior venocaval obstruction secondary to central venous catheter-related thromboses in two patients with metastatic colorectal carcinoma receiving weekly 5-flurouracil. Should adjusted-dose warfarin be used as thromboprophylaxis?

Standard treatment for patients with metastatic colorectal carcinoma (MCC) involves treatment with weekly 5-flurouracil (5-FU) chemotherapy by continuous infusion, requiring the insertion of a central venous catheter (CVC). One of the main complications of CVCs is venous thromboembolic disease (VTE), with an incidence varying between 3 and 54% in different studies. During the past 14 months, 17 patients with MCC have been treated weekly with 5-FU in our unit, comprising 11 males and six females with a mean age of 60 years (range, 49-72 years). Thromboprophylaxis for all patients included 1 mg/day warfarin unless contraindicated. Three patients developed venography-confirmed CVC-related VTE, including two cases of occlusive superior venocaval VTE. All three patients were treated with intravenous thrombolytic drugs; two responded completely and one partially. We wonder whether the incidence of CVC-related VTE may be reduced further by using adjusted-dose warfarin rather than fixed low-dose warfarin. However, one has to be guarded because of the greater risk of bleeding with more intensive anticoagulation, especially in patients with liver metastases. Ongoing studies such as the warfarin prophylaxis study are essential to determine the safety and efficacy of different approaches in order to determine the optimum thromboprophylaxis for this group of patients.

Electrospray tandem mass spectrometry for analysis of acylcarnitines in dried postmortem blood specimens collected at autopsy from infants with unexplained cause of death.

BACKGROUND: Deaths from inherited metabolic disorders may remain undiagnosed after postmortem examination and may be classified as sudden infant death syndrome. Tandem mass spectrometry (MS/MS) may reveal disorders of fatty acid oxidation in deaths of previously unknown cause. METHODS: We obtained filter-paper blood from 7058 infants from United States and Canadian Medical Examiners. Acylcarnitine and amino acid profiles were obtained by MS/MS. Specialized interpretation was used to evaluate profiles for disorders of fatty acid, organic acid, and amino acid metabolism. The analyses of postmortem blood specimens were compared with the analyses of bile specimens, newborn blood specimens, and specimens obtained from older infants at risk for metabolic disorders. RESULTS: Results on 66 specimens suggested diagnoses of metabolic disorders. The most frequently detected disorders were medium-chain and very-long-chain acyl-CoA dehydrogenase deficiencies (23 and 9 cases, respectively), glutaric acidemia type I and II deficiencies (3 and 8 cases, respectively), carnitine palmitoyl transferase type II/translocase deficiencies (6 cases), severe carnitine deficiency (4 cases), isovaleric acidemia/2-methylbutyryl-CoA dehydrogenase deficiencies (4 cases), and long-chain hydroxyacyl-CoA dehydrogenase/trifunctional protein deficiencies (4 cases). CONCLUSIONS: Postmortem metabolic screening can explain deaths in infants and children and provide estimates of the number of infant deaths attributable to inborn errors of metabolism. MS/MS is cost-effective for analysis of postmortem specimens and should be considered for routine use by Medical Examiners and pathologists in unexpected/unknown infant and child death.

Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.

The circadian clock mutation alters sleep homeostasis in the mouse.

The onset and duration of sleep are thought to be primarily under the control of a homeostatic mechanism affected by previous periods of wake and sleep and a circadian timing mechanism that partitions wake and sleep into different portions of the day and night. The mouse Clock mutation induces pronounced changes in overall circadian organization. We sought to determine whether this genetic disruption of circadian timing would affect sleep homeostasis. The Clock mutation affected a number of sleep parameters during entrainment to a 12 hr light/dark (LD 12:12) cycle, when animals were free-running in constant darkness (DD), and during recovery from 6 hr of sleep deprivation in LD 12:12. In particular, in LD 12:12, heterozygous and homozygous Clock mutants slept, respectively, approximately 1 and approximately 2 hr less than wild-type mice, and they had 25 and 51% smaller increases in rapid eye movement (REM) sleep during 24 hr recovery, respectively, than wild-type mice. The effects of the mutation on sleep are not readily attributable to differential entrainment to LD 12:12 because the baseline sleep differences between genotypes were also present when animals were free-running in DD. These results indicate that genetic alterations of the circadian clock system and/or its regulatory genes are likely to have widespread effects on a variety of sleep and wake parameters, including the homeostatic regulation of sleep.

Synthesis and SAR of benzyl and phenoxymethylene oxadiazole benzenesulfonamides as selective beta3 adrenergic receptor agonist antiobesity agents.

Benzyl and phenoxymethylene substituted oxadiazoles are potent and orally bioavailable beta3 adrenergic receptor (AR) agonists. The 4-trifluormethoxy substituted 5-benzyl oxadiazole 5f has an EC50 of 8 nM in the beta3 AR agonist assay with 100-fold selectivity over beta1 and beta2 AR binding inhibition activity. Its oral bioavailability in dogs is 30 +/- 4%, with a half-life of 3.8 +/- 0.4 h. In the anesthetized rhesus, 5f evoked a dose-dependent glycerolemia (ED50Gly = 0.15 mg/kg). Under these conditions a heart rate increase of 15% was observed at a dose level of 10 mg/kg.

Orientation of Phalerisida maculata Kulzer (Coleoptera, Tenebrionidae) in sandy beaches of the Chilean coast. Orientation of Phalerisida maculata in sandy beaches.

The orientation stimuli of adult individuals of the beetle Phalerisida maculata Kulzer (Coleoptera, Tenebrionidae) over the beach surface, were studied at two sandy beaches of the chilean coast approximately 1300 km apart, Apolillado (ca. 29 degrees S) and Playa Universitaria de Mehuín (ca. 39 degrees S). Phalerisida maculata did not orient by astronomic cues such as the sun and moon, nor by the terrestrial magnetic field. Both populations showed positive scototaxis, and oriented downward on slopes with dry sediments, and upwards on slopes with wet sediments.

Daily social and physical activity increases slow-wave sleep and daytime neuropsychological performance in the elderly.

Decreased levels of physical and social activity associated with aging can be particularly pronounced in residents of assisted living facilities. Reduced exposure to important behavioral and time-giving cues may contribute to the age-related changes in circadian rhythmicity and sleep. The present study was conducted to test the hypothesis that an enforced schedule of structured social and physical activity (0:900 to 10:30 and 19:00 to 20:30 daily for two weeks) can have beneficial effects on circadian rhythmicity, nocturnal sleep, daytime functioning, mood, and vigor. The subjects were 14 elderly residents of continued-care retirement facilities while a similar group of 9 elderly residents served as controls. The group exposed to structured activities had increased amounts of slow-wave sleep and demonstrated improvement in memory-oriented tasks following the intervention. Conversely, no significant changes were noted in the amplitude and phase of the body temperature rhythm or in subjective measures of vigor and mood. These results indicate that short-term exposure to structured social intervention and light physical activity can significantly improve memory performance and enhance slow-wave sleep in older adults without alterations to the circadian phase or amplitude of body temperature. This is the first report to demonstrate that low intensity activity in an elderly population can increase deep sleep and improve memory functioning. The high degree of interest in these activities paired with the simple nature of the tasks makes this a potentially practical intervention which can be adapted for both community dwelling and assisted-living elders.

Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid, and amino acid metabolism.

Development of acylcarnitine and amino acid profiling using tandem mass spectrometry, and its application for use with dried blood specimens collected on filter-paper cards, has introduced an innovative new technology for detecting inborn errors of fatty acid, organic acid, and amino acid metabolism. From November 1, 1992 through June 30, 1999 we screened more than 700,000 newborns in Pennsylvania, Ohio, North Carolina, and Louisiana. We have prospectively detected 163 inborn errors of metabolism. Eighty-six patients have amino acid metabolism errors. Among them are phenylketonuria, hyperphenylalaninemia, maple syrup urine disease, and several urea cycle disorders. Thirty-two have organic acid metabolism errors, including glutaric aciduria type 1; 3-methylcrotonyl coenzyme A (CoA) carboxylase deficiency, propionic acidemia, methylmalonic acidemia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; and 45 have fatty acid oxidation errors, including 36 with medium-chain acyl-CoA dehydrogenase deficiency. Details of the methodology are presented and the potential of this screening technology is discussed.

Optimization of an automated DNA purification protocol for neonatal screening.

CONTEXT: Collection of blood from newborns is a standard clinical procedure used for genetic screening. Typically, blood from a heel prick is absorbed onto standard collection paper and dried before analysis of metabolites, proteins, hormones, and more recently DNA. OBJECTIVE: To evaluate strategies to purify DNA for use with automated workstations. DESIGN: Two factors were used to evaluate several DNA purification protocols: residual heme contamination and amplification yield. The protocol that produced DNA with the lowest heme content and the highest amplification yield was selected. In combination with those two performance factors, the protocol with the fewest number of steps was chosen to reduce reagent use and processing time. SETTING: Industrial research and development laboratory. RESULTS: Robust amplification of DNA isolated from dried blood spots was demonstrated using both fluorescence and agarose gel-based detection methods. In addition, the samples had consistent DNA volumes and had no detectable cross-contamination. Suggested instrument settings, equipment, and supplies were included for automated processing of DNA from dried blood spots. CONCLUSION: A 4-step DNA processing protocol was developed for dried blood spots. The protocol could be performed in either a manual or automated format, making it possible to process hundreds of samples in 1 day.

Immunoreactive trypsinogen levels in pediatric patients with intestinal failure awaiting intestinal transplantation.

The aim of this study was to evaluate pancreatic function in total parenteral nutrition (TPN)-dependent children with permanent intestinal failure by measuring immunoreactive trypsinogen (IRT) levels. Between 1992 and 1996, 105 pediatric patients with permanent intestinal failure were referred to the Children's Hospital of Pittsburgh for small intestinal transplant evaluation. Serum samples were available from 55 of them. Ten suffered from intestinal pseudo-obstruction or microvillus inclusion disease, while 45 had short bowel syndrome (SBS). IRT levels were significantly higher (p < 0.001) in SBS patients (89.4 +/- 9.2 ng mL) compared to controls (43.4 +/- 5.6 ng/ nL) without liver, gastrointestinal, or kidney disease. IRT levels did not correlate with liver injury, length of bowel, or the cause of SBS. Five of 20 patients who underwent intestinal transplantation developed pancreatitis during a median post-operative follow up 15.4 months later. IRT levels failed to predict who would develop pancreatitis post-transplant. The data suggest that elevated plasma IRT levels are common among children with intestinal failure, but fail to identify patients at risk for pancreatitis post-transplant.

Human beta3 adrenergic receptor agonists containing cyclic ureidobenzenesulfonamides.

Human beta3 adrenergic receptor agonists containing 5-membered ring ureas were shown to be potent partial agonists with excellent selectivity over beta1 and beta2 binding. L-760,087 (4a) and L-764,646 (5a) (beta3 EC50 = 18 and 14 nM, respectively) stimulate lipolysis in rhesus monkeys (ED50 = 0.2 and 0.1 mg/kg, respectively) with minimal effects on heart rate. Oral absorption in dogs is improved over other urea analogs.