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Aims: Present-day screening of the diabetic foot involves the Semmes Weinstein Monofilament Test for evaluating loss of sensibility, while testing for intrinsic muscle weakness is not implied. Just as with the early detection of sensibility loss, early detection of intrinsic muscle weakness might have important implications for the prevention of both ulceration and deformity in patients with diabetes. The purpose of this study is to investigate the prevalence of patients with diabetes presenting intrinsic muscle weakness of the hallux, but with a normal sensibility of the sole of the foot.Methods: A cross-sectional study design was applied. Intrinsic muscle function of the hallux was measured with the Paper Grip Test, while sensibility of the sole of the foot was measured with the Semmes Weinstein Monofilament Test 5.07/10-g.Results: In a period of three months a total of 266 patients with diabetes (mean age 60, 134 females (50%), 177 type 2 diabetes mellitus (67%)) met the inclusion criteria and were examined for both intrinsic muscle weakness of the hallux and sensibility of the soles of the feet. The results showed that intrinsic muscle weakness was present more frequent in patients with impaired sensibility (P = 0.001), also 20% of the population had intrinsic muscle weakness in the presence of normal sensibility. Multivariate regression analysis showed that only age is associated with patients with diabetes presenting normal sensibility but impaired intrinsic muscle function (P = 0.017).Conclusions: The Paper Grip Test could have added value to current physical examination of the feet in patients with diabetes. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Background/Aims: Accumulating evidence from a relatively small number of prospective studies indicates that exposure to prenatal stress profoundly influences the developing human fetus with consequences that persist into childhood and very likely forever. Methods: Maternal/fetal dyads are assessed at ∼20, ∼25, ∼31 and ∼36 weeks of gestation. Infant assessments begin 24 h after delivery with the collection of cortisol and behavioral responses to the painful stress of the heel-stick procedure and measures of neonatal neuromuscular maturity. Infant cognitive, neuromotor development, stress and emotional regulation are evaluated at 3, 6 12 and 24 months of age. Maternal psychosocial stress and demographic information is collected in parallel with infant assessments. Child neurodevelopment is assessed with cognitive tests, measures of adjustment and brain imaging between 5 and 8 years of age. Results:Psychobiological markers of stress during pregnancy, especially early in gestation, result in delayed fetal maturation, disrupted emotional regulation and impaired cognitive performance during infancy and decreased brain volume in areas associated with learning and memory in 6- to 8-year-old children. We review findings from our projects that maternal endocrine alterations that accompany pregnancy and influence fetal/infant/child development are associated with decreased affective responses to stress, altered memory function and increased risk for postpartum depression. Conclusions: Our findings indicate that the mother and her fetus both are influenced by exposure to psychosocial and biological stress. The findings that fetal and maternal programming occur in parallel may have important implications for long-term child development and mother/child interactions.

Background: Developing countries are facing a rise in noncommunicable diseases (NCD), which is a cause for concern. The World Health Organization has recommended a stepwise approach for NCD risk factor surveillance. Screening for risk factors in remote populations is difficult due to lack of resources and technical expertise, including standardized laboratory facilities. The collection of samples on filter paper for the assessment of risk factors circumvents the need for blood processing, storage, and shipment at ultralow temperatures. Method: Samples were collected on 3-mm Whatman filter paper from one industry (National Thermal Power Corporation) located in the periphery of Delhi as part of a surveillance carried out in industries from different parts of India. Total cholesterol was measured in serum and dried blood by the cholesterol oxidase/p-aminophenazone method and triglycerides by the glycerophosphate oxidase–peroxidase/aminophenazone method. Values obtained by the two methods were compared using Pearson correlation, and Bland–Altman plots were prepared to assess bias. Results: The correlation coefficient “ r” was 0.78 for cholesterol and 0.94 for triglycerides between dried blood spots and serum. Bland–Altman plots suggest that differences in values obtained by the two methods were within two standard deviations for most of the samples. Conclusions: Blood samples dried on filter paper can be a successful option for population screening in remote areas, provided preanalytical variations arising due to the method of blood spot preparation and storage are well controlled.

Although the genetic basis of human sexual determination and differentiation has advanced considerably in recent years, the fact remains that in most subjects with disorders of sex development (DSD) the underlying genetic cause is unknown. Where pathogenic mutations have been identified, the phenotype can be highly variable, even within families, suggesting that other genetic variants are influencing the expression of the phenotype. This situation is likely to change, as more powerful and affordable tools become widely available for detailed genetic analyses. Here, we describe recent advances in comparative genomic hybridisation, sequencing by hybridisation and next generation sequencing, and we describe how these technologies will have an impact on our understanding of the genetic causes of DSD.

To evaluate the performance of dried blood spots (DBSs) with subsequent analyses of glutamic acid decarboxylase (GADA) and islet antigen-2 (IA-2A) with the RSR-ELISAs, we selected 80 children newly diagnosed with type 1 diabetes and 120 healthy women. DBSs from patients and controls were used for RSR-ELISAs while patients samples were analysed also with in-house RIAs. The RSR-ELISA-GADA performed well with a specificity of 100%, albeit sensitivity (46%) was lower compared to in RIA (56%;P=.008). No prozone effect was observed after dilution of discrepant samples. RSR-ELISA-IA-2A achieved specificity of 69% and sensitivity was lower (59%) compared with RIA (66%;P<.001). Negative or low positive patients and control samples in the RSR-ELISA-IA-2A increased after dilution. Eluates from DBS can readily be used to analyse GADA with the RSR-ELISA, even if low levels of autoantibodies were not detected. Some factor could disturb RSR-ELISA-IA-2A analyses.

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be ‘reversed’ by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90α, HSP90β, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90α (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed.

Screening for congenital hypothyroidism for all newborns of the former western parts of the city of Berlin was instituted in 1978 by determination of the TSH levels in dried filter paper blood spots of the 3rd to 5th day of life. Since 1991 the newborns of the former eastern parts have been also included in the same screening program. From 1978 to 1995 a total number of 104 newborns with permanent congenital hypothyroidism have been detected resulting in a prevalence of congenital hypothyroidism of 1:3800. The etiological diagnosis of hypothyroidism was made by imaging studies (ultrasonography or 99mTc scintigraphy) and assessment of serum thyroglobulin and thyroid hormone levels. Using this approach in 37 children (30 female, 7 male) the diagnosis of athyrosis, in 20 children (15 female, 5 male) the diagnosis of ectopy and in 21 children (18 female, 3 male) the diagnosis of thyroid hypoplasia was made, 16 children (8 female, 8 male) had a normally sized gland and 4 (1 female, 3 male) had congenital goiter. In 86% of all patients the age at onset of thyroxine (L-T4) replacement therapy was 8 or 9 days of life (3-42 days) and the median initial L-T4 dose was 14 micrograms/kg/day (10-16 micrograms/kg/day). The intellectual outcome of 77 children (2-16 years) was studied and normal scores for the intelligence (IQ) and developmental (DQ) quotients were found in 71 (92%). Outcome was not correlated to the age at onset of treatment, the initial dose and the severity of hypothyroidism, but there was a positive correlation of the socioeconomic status of the family and the IQ of the patients. The results of the screening program in Berlin document that an early and efficient thyroxine replacement can normalize the intellectual outcome of patients with congenital hypothyroidism independent of the severity of the disease as assessed by the residual thyroid function detectable at diagnosis.

Insulin-like growth factor-I (IGF-I) is sensitive to nutritional stress and is reduced in soldiers during stressful field training. Methods have recently been developed to measure IGF-I from filter paper blood spots. Filter paper has advantages over traditional blood sampling in that neither blood separation equipment nor refrigeration is necessary after sample collection. This study determined whether filter paper blood spots collected in a field environment could measure IGF-I and subsequent changes during military operational stress. Thirty-four Marines participating in an 8-day military field exercise characterized by near-continuous physical work (total daily energy expenditure 17-25 MJ/day) and underfeeding (dietary intake 7.0 MJ/day) had blood samples taken on day 0, day 4, and day 8. IGF-I was measured by filter paper blood spot assays from fingertip blood samples and by conventional methods using serum. Correlation and measurement agreement were assessed. Blood spot (Day 0 152 +/- 6 ng mL(-1)Day 4 111 +/- 6 ng mL(-1)Day 8 74 +/- 4 ng mL(-1)) and serum IGF-I (Day 0 412 +/- 10 ng mL(-1)Day 4 258 +/- 14 ng mL(-1)Day 8 203 +/- 13 ng mL(-1)) concentrations declined (p0.05) progressively over the 8-day exercise. Overall, the two methods significantly (p0.05) correlated (r = 0.92); however, the blood spot values were on average 61% lower than serum, but could be used to predict serum values ( +/- 10%). IGF-I is a biomarker of metabolic status. The filter paper blood spot method for IGF-I detected reductions accompanying nutritional stress and may be of potential value for characterizing the IGF-I response when conventional blood sampling methods are not feasible.

Background Krabbe disease (KD) is an inherited lysosomal storage disease (LSD) caused by the deficiency of galactocerebrosidase (GALC) and is characterized by a severe and progressive leukodystrophy with death frequently before one year of life in the classical early-onset form. As a consequence of the enzyme defect, globoid cells containing undigested galactosylceramide are observed and are characteristic of the disease. Hematopoietic stem cell transplantation is the current treatment for this disease, with some success in the classical cases if performed very early in life. Definitive diagnosis of KD is generally accessed by determination of GALC in leukocytes or fibroblasts. For the last few years, dried-blood filter paper (DBFP) samples have been increasingly used for lysosomal enzyme assays. Originally, some lysosomal enzymes could not be tested in DBFP samples using fluorometric assays, including GALC, heparan-sulfamidase and a few others. Recently, we reported successful results using dried-leukocytes filter paper (DLFP) samples for heparan sulfamidase and β-galactosidase. Extending these studies, we present now a new GALC assay on these type of samples. Methods Adapted leukocyte fluorometric assay was used for the evaluation of GALC in DLFP samples. Results Our results using this method showed a clear discrimination between GALC levels observed in KD patients and healthy controls. Conclusions The assay is robust and reliable and could be adopted by reference laboratories for diagnosis of LSDs. It is expected that the use of DLPF would make it possible to diagnose patients living in isolated areas, where liquid samples usually have to be transported over several days and sometimes across country borders before reaching reference laboratories.

Classic CAH (salt-wasting and simple virilizing) meets all of the recommended criteria for newborn screening. There are reliable and efficient newborn screening tests, the disorder results in high morbidity and mortality if left undetected, there is effective treatment that reduces negative outcomes, and there is a relatively high incidence. When compared with the case findings without the benefit of screening, the data from screening programs show reduced adrenal crises, reduced incorrect sex assignments, and reduced deaths. Racial/ethnic prevalence differences are present in newborn screening program data. The Texas data indicate a lower disease frequency in African-Americans when compared with Caucasians, and international data indicate higher frequencies in native Yupik Eskimos, Brazilians, residents of La Reunion, and Filipinos. When worldwide clinical ascertainment data are compared with newborn screenng data, it is clear that newborns with CAH (especially males) die when screening is not done. To be effective in reducing mortality, newborn screening must be performed soon after birth, and the results must be available quickly so that early salt-wasting crises can be averted. It is preferable that newborn screening laboratiories be operational 7 days a week, and that sample delivery from the collection site to testing laboratory be as efficient as possible, including weekends and holidays, so that undue testing delays are not encountered. These two requirements pose major challenges for most programs, but they are critical to optimal screening outcome. Based on the studies in Texas, with second screening samples collected at approximately 2 weeks of age, some newborns with simple virilizing CAH are missed on initial screening using current testing protocols. There is need to set a screening cut-off such that the false-positive rate does not oversaturate the follow-up system, in part owing to the insensitivity of current kit methodologies and the biochemical manifestations of CAH. With advances in genetic testing procedures and improved automation techniques, it may soon be possible for CAH screening programs to include genotyping as a second-tier confirmation as a part of the newborn screening protocol. Despite the fact that CAH is a continuum of disorders, the correlation between genotype and phenotype is fairly consistent in most cases. For the purpose of screening, genotyping will likely be useful only for differential diagnoses of non-salt wasters, given the necessary time constraints and expense of obtaining genotypes and the need for immediate diagnosis/treatment of salt wasters. It is hoped that newborn screening programs will begin to provide answers to some of these question in addition to their primary function of reducing the morbidity and mortality resulting from CAH.

The measurement of insulin-like growth factor I (IGF-I) from extracted whole dried blood spots on filter paper is a simple method allowing sample collection to be carried out by the patient at home. One drop of blood is applied to a filter card, air dried and posted to the laboratory. The IGF-I is dissociated from its binding protein by an acidification and neutralization procedure, and IGF-I is then measured by an enzyme-linked immunosorbent assay. Results from the analysis of IGF-I using blood spots on filter paper compared favourably with those from conventional methods, thus showing it to be an ideal and cost-effective technique for the measurement of IGF-I in both adults and children.

OBJECTIVE To facilitate HbA1c determination, we evaluated an HbA1c filter paper system enabling capillary blood sampling at home by the patients. RESEARCH DESIGN AND METHODS Capillary blood (two drops) was applied to a filter paper (HbA1c Via Post) and sent to the laboratory where a small disc was punched out on the filter paper. Hemoglobin was eluted from the disc in a buffer containing cysteine to eliminate the interfering glutathione adduct (HbA3) formed during storage. Analysis was performed by ion-exchange chromatography (Mono S, high-performance liquid chromatography), and the eluate was compared with hemolysate of venous blood from 41 patients. The stability of blood impregnated on filter paper was checked at different temperatures over different periods of time. RESULTS There was an excellent agreement (r = 0.99) between HbA1c values from capillary blood on filter paper and HbA1c values from venous blood. HbA1c values were constant when stored on filter paper for 5–7 days at 20–21°C (room temperature) or at 4–6°C (refrigerator) for 10 days as well as at −70°C for several months after blood sampling. A new chromatographic-interfering hemoglobin fraction both from venous and capillary samples was identified as free alpha-chain of hemoglobin. CONCLUSIONS The HbA1c filter paper system enables capillary blood sampling at home, eliminates the need of vein puncture in children and adults, and provides the diabetologist with an HbA1c value when the patient visits the clinic without a need for a previsit phlebotomy.

Background: A large number of nondigitized electrocardiograph (ECG) strips are routinely collected in larger cohort studies such as the ADDITION study (Anglo-Danish-Dutch Study of Intensive Treatment in People with Screen-Detected Diabetes in Primary Care). These ECG strips are routinely read manually but may contain overlooked information revealing cardiac autonomic dysfunction. The aim of this study was to investigate whether clinical information may be lost using manual R wave to R wave (RR) interval measurements in the calculation of heart rate variability (HRV) in patients with type 2 diabetes mellitus (T2DM). Method: From the Danish part of the ADDITION study, we randomly selected 120 T2DM patients at baseline of the ADDITION study. Analysis of the ECG strips was performed using two different methods: (1) by experienced technicians using rulers and (2) by experienced technicians using a high-resolution computer-assisted method. Calculation of heart rate and time domain HRV [standard deviation of normal-to-normal RR intervals (SDNN) and root mean square of successive differences (RMSSD)] were performed with the same software. Results: When comparing results from the two methods, the following values of Pearson's r are obtained: 0.98 for heart rate, 0.76 for SDNN, and 0.68 for RMSSD. These results indicate that heart rate and HRV measurements by the computer-assisted and manually based methods correlate. However, Bland-Altman plots and Pitman's test of difference in variance revealed poor agreements ( p < .01) for both HRV measurements (SDNN and RMSSD); only heart rate showed substantiated agreement ( p = .54) between the two methods. Low HRV was statistically significantly associated to high heart rate, systolic blood pressure, and diastolic blood pressure in these screen-detected T2DM patients. Conclusions: Paper ECG strips may contain overlooked clinical information on the status of autonomic function in patients with T2DM. In our study, manual measurements of RR intervals were inferior to the computer-assisted method. Based on this study, we recommend cautiousness in the clinical use and interpretation of HRV based on manual or low resolution measurements of RR intervals from ECG strips. High resolution measurements of RR intervals reveal significant associations between low HRV and high heart rate, systolic blood pressure, and diastolic blood pressure among patients with screen-detected T2DM. It is feasible to use a computer-assisted method to determine RR intervals in patients with T2DM.

The molecular steps in normal sexual development were largely discovered by studying patients and animal models with disorders of sexual development (DSD). Although several types of DSD have been reported in the cat and dog, which are often strikingly similar to human DSD, these have been infrequently utilized to contribute to our knowledge of mammalian sexual development. Canine and feline cases of DSD with sufficient evidence to be considered as potential models are summarized in this report. The consensus DSD terminology, and reference to previous terminology, is used to foster adoption of a common nomenclature that will facilitate communication and collaboration between veterinarians, physicians, and researchers. To efficiently utilize these unique resources as molecular tools continue to improve, it will be helpful to deposit samples from valuable cases into repositories where they are available to contribute to our understanding of sexual development, and thus improve human and animal health.

Fetal glucocorticoid exposure is a key mechanism proposed to underlie prenatal ‘programming’ of adult affective behaviours such as depression and anxiety. Indeed, the glucocorticoid metabolising enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which is highly expressed in the placenta and the developing fetus, acts as a protective barrier from the high maternal glucocorticoids which may alter developmental trajectories. The programmed changes resulting from maternal stress or bypass or from the inhibition of 11β-HSD2 are frequently associated with alterations in the hypothalamic-pituitary-adrenal (HPA) axis. Hence, circulating glucocorticoid levels are increased either basally or in response to stress accompanied by CNS region-specific modulations in the expression of both corticosteroid receptors (mineralocorticoid and glucocorticoid receptors). Furthermore, early-life glucocorticoid exposure also affects serotonergic and catecholamine pathways within the brain, with changes in both associated neurotransmitters and receptors. Indeed, global removal of 11β-HSD2, an enzyme that inactivates glucocorticoids, increases anxiety- and depressive-like behaviour in mice; however, in this case the phenotype is not accompanied by overt perturbation in the HPA axis but, intriguingly, alterations in serotonergic and catecholamine pathways are maintained in this programming model. This review addresses one of the potential adverse effects of glucocorticoid overexposure in utero, i.e. increased incidence of affective behaviours, and the mechanisms underlying these behaviours including alteration of the HPA axis and serotonergic and catecholamine pathways.

One source of deoxyribonucleic acid (DNA) for genetic studies is the utilization of dried blood spots stored on paper cards (Guthrie cards) collected shortly after birth. These cards represent an important source of material for epidemiologic and population-based genetic studies. Extraction of DNA from these cards can lead to variable amounts of recovered DNA. We report here results of our efforts to maximize yield from this valuable, but nonrenewable, resource.Commercial methods of DNA extraction from blood cards were used, and protocol modifications were introduced that enhanced DNA yield.Use of a commercial solvent prior to DNA extraction steps gave greater yields than extraction without the solvent. Modification of the elution step by use of prewarmed extraction buffer and a soaking step at an elevated temperature increased yield by 6- to 10-fold.The modified DNA extraction method yielded as much as 660 ng of DNA from a single 5-mm-diameter punch of a blood spot card. The DNA performed well in downstream, polymerase chain reaction-based applications.

Geckos are a species-rich clade of reptiles possessing diverse sex determining mechanisms. Some species possess genetic sex determination, with both male and female heterogamety, while other species have temperature-dependent sex determination. I compiled information from the literature on the taxonomic distribution of these sex determining mechanisms in geckos. Using phylogenetic data from the literature, I reconstructed the minimum number of transitions among these sex determining mechanisms with parsimony-based ancestral state reconstruction. While only a small number of gecko species have been characterized, numerous changes among sex determining mechanisms were inferred. This diversity, coupled with the high frequency of transitions, makes geckos excellent candidates as a model clade for the study of vertebrate sex determination and evolution.

Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin βA subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin α and βB subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility.

Adaptation of assays on dried blood has advantages of ease of collection, transportation, minimal invasiveness and requirement of small volume. A method for extraction and estimation of triglyceride from blood spots dried on filter paper (Whatman no. 3) has been developed. A single dried blood spot containing 10 μL blood was used. Triglyceride was efficiently extracted in methanol from blood dried on filter paper by incubation at 37°C for two hours with gentle shaking. For the estimation, a commercially available enzymatic method was used. Blood spot assays showed mean intra and inter assay coefficient of variance of 6.0% and 7.4% respectively. A comparison of paired whole blood spots and plasma samples (n = 75, day 0) gave an intraclass correlation of 0.96. The recovery was 99.6%. The dried blood triglyceride concentrations were stable for one month when the filter discs were stored at room temperature (16–28°C). Storage of filters at 4°C extended the stability and triglycerides could be quantatively recovered after 3 months of storage.

Objective –To compare glycosylated hemoglobin (GH) results obtained by filter paper fingerstick collection and mailed for assay by affinity chromatography with results from a venous sample assayed by ion-exchange chromatography (HbA1) in a local laboratory. Research Design and Methods –Fifty-eight volunteer subjects with insulin-dependent diabetes mellitus (IDDM), aged 5-24 yr, included patients at a referral-based IDDM clinic and subjects in an ongoing research study. We obtained two blood samples from each subject. One was collected by fingerstick onto filter paper, the other by venipuncture into a vacutainer. We sent filter paper samples to the Diabetes Research Laboratory (Univ. of Missouri, Columbia, MO) for analysis. Vacutainer samples were sent to the Clinical Chemistry Department of the Clinical Laboratory, University of Colorado Health Sciences Center. Results –Results were highly correlated (r = 0.89, P = 0.0001). Fifty-nine percent were classified identically when results were normalized to SD units and grouped to suggest levels of clinical concern. Conclusions –The filter paper method is a convenient, accurate measure of glycosylated hemoglobin in young people with IDDM. It should be considered a credible alternative research and clinical tool.

A simple and reliable method of paired TSH assay was developed and used in screening for neonatal primary hypothyroidism. In this method, a paired assay is first done. Equal parts of the extracts of dried blood spots on filter paper (9 mm diameter) from two infants 4-7 days old are combined and assayed for TSH by double antibody RIA. If the value obtained is over the cut-off point, the extracts are assayed separately for TSH in a second assay to identify the abnormal sample. Two systems, A and B, with different cut-off points were tested. On the basis of reference blood samples (serum levels of TSH, 80 microU/ml in system A and 40 microU/ml in system B), the cut-off point was selected as follows: upper 5 (A) or 4 (B) percentile in the paired assay and values of reference blood samples in the second individual assay. Four cases (2 in A and 2 in B) of neonatal primary hypothyroidism were found among 25 infants (23 in A and 2 in B) who were recalled from a general population 41,400 infants (24,200 in A and 17,200 in B) by 22,700 assays. This paired TSH neonatal hypothyroidism.

We have simultaneously measured T4 and TSH concentrations from 93,000 consecutive filter paper blood samples received by the Quebec network for Genetic Medicine. T4 was measured using the Micromedic Automated System and TSH was measured by RIA kits from Pharmacia and Becton Dickinson, each for a 6-month period. Statistical analyses to assess quality control parameters for the various methods revealed that the T4 assay had greater precision and reproducibility than either of the 2 commercially available TSH kits. The number of false negative results was similar for both methods. Eight infants with ectopic thyroid glands were correctly detected by the T4 method. One infant with secondary hypothyroidism was detected by the T4 approach and missed by the TSH methodology. Three infants overall would have been missed using either the T4 or TSH approach. These data indicate that primary T4 screening has similar sensitivity compared to TSH kits for mass screening programs for congenital hypothyroidism.

A pilot newborn screening program for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) was conducted in Alaska using a 3-mm disc filter paper elution technique of capillary whole blood for 17-hydroxyprogesterone (17-OHP) by RIA. The filter paper specimens were provided via the mandated newborn screening program. The highest values of 17-OHP in a control population of 4569 consecutive normal neonates, aged 2–14 days, was 40 pg/disc, and the range of values for 20 newborns with proven CAH was 57–980 pg/disc. All Alaskan newborns with 17-OHP levels of 57 pg/disc or greater were referred for diagnostic workup, and initially, those with 17-OHP levels of 41–56 pg/disc were recalled for a second specimen. However, the range for recall was changed subsequently to the 17-OHP level greater than 51 pg/disc, since no newborns with 17-OHP level below 50 pg/disc were found to be affected in the initial screening. In a 30-month period, the Alaskan newborns screened on the third day of life consisted of a t...

It would be advantageous to be able to measure glycosylated hemoglobin (GHb) from a sample of blood dried on paper and sent to the physician by the patient. Indeed, such a technique has been introduced by Isolab (Akron, OH). We tested the validity of the method to determine whether 1) applying the blood to paper and immediately assaying it correlates with the same sample assayed by the usual wholeblood technique, 2) the size of sample applied to paper affects apparent GHb results, 3) time and temperature of storage of the sample paper affect apparent GHb resu ts, and 4) plasma glucose concentration of the sample affects GHb results. The GHb of samples assayed immediately after application to sample paper versus those assayed as whole blood showed very good correlation (r = .93, P < .001). Volume of the drop of blood applied to paper (25–100 μl) did not affect results. However, there was a dramatic, temperature-dependent increase in apparent GHb when samples were stored on sample paper, averaging 12.5, 16.3, and 19.5% when stored at room temperature for 3, 6, and 9 days, respectively. Overall, apparent GHb rose from 1.3-fold in 3 days at 4°C to 3.8-fold in 9 days at 37°C. The rate of GHb formation was proportional to plasma glucose concentration, but removal of free glucose by ethanol or glucose oxidase did not yield consistent results for this method. We conclude that these sample papers are not useful as an approach to collecting blood samples for GHb measurement.

The rate of urea formation in rat liver slices was retarded when any of the following metabolites was added to the incubation medium: glucose, fructose, lactate, pyruvate or succinate. Acetate and octonoate were without effect. Pyruvate appeared to be the most potent of the inhibitors tested. Attempts to elucidate the mechanism of the pyruvate effect revealed that the inhibition was substrate dependent and could be offset by an excess of ornithine. This and other evidence was thought to suggest that pyruvate might act by transaminating with ornithine, thereby interrupting urea synthesis by siphoning off this “catalytic” intermediate of the urea cycle. The results suggest that one of the biochemical events following carbohydrate intake might be a direct, hormone-independent interaction between the intermediary metabolism of carbohydrate and the process of urea synthesis. The possible relationship between these observations and the protein-sparing action of carbohydrates is discussed.

The method of Haber for the purification of (125I)-labeled angiotensin by high volt age paper electrophoresis has been modified. Instead of eluting the labeled peptide from the paper in a single bulk elution, serial small elutions are collected, resulting in a highly purified product in later elutions.

A new sensitive radioimmunoassay method for measuring reverse triiodothyronine (rT 3 ) concentrations in dried blood samples, designed to screen newborn infants for congenital hypothyroidism, has been developed. Paper strips are impregnated with cord blood and dried. Duplicate 5-mm diameter discs are punched from the paper strips and added directly to the radioimmunoassay reaction mixture. After incubation, bound and free hormone are separated by dextran-coated charcoal. The disc remains in the solution throughout the procedure and the assay can be completed within 24 hr. Recovery of rT 3 is greater than 95% and coefficients of variation are 9.4% (intraassay) and 12.2% (interassay) at an rT 3 concentration of 220 ng/dl. At very low rT 3 concentrations (25 ng/dl), coefficients of variation are 14.2% (intraassay) and 18.7% (interassay). The method readily detects 12.5 ng/dl of rT 3 . With this paper disc method, rT 3 was measured in 38 newborns and compared with serum rT 3 measured in the same subjects by a standard radioimmunoassay method. The correlation between rT 3 values measured in dried blood disc and in serum was very high ( r = 0.918). The rT 3 in dried blood discs from the cord blood of 745 normal newborns was 228.9 ± 76.0 ng/dl (mean ± SD). In contrast, two infants with proven congenital hypothyroidism had rT 3 values of 35 and 75 ng/dl, respectively. This study indicates that rT 3 can be easily measured in dried blood discs and suggests that the described method may be a useful screening procedure in a program for the detection of neonatal hypothyroidism.

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.

131I-insulin was given intravenously to normal rats, and the passage of radioactivity into the duodenal loop containing a proteasis inhibitor was studied by two techniques. In the first, the 131I-insulin was separated from the intestinal content by electrophoresis; whereas in the second, the radioactivity that remained on an ion-exchange resin, directly introduced into the small intestine, was measured. It was found that within 15 minutes from the injection a small amount of 131I-insulin passes into the gut lumen. — The absorption of 131I-insulin from small intestine into the blood was studied after introduction of 131I-insulin along with a proteasis inhibitor into the intestinal loop. The autoradiograph of the electrophoresis of serum showed plasma-protein-bound radioactivity but no radioactivity due to unbound (free) insulin-131I. — These findings show that the passage of insulin through the intestinal wall of rats can be demonstrated by neutralizing the proteolytic activity in the intestinal tract. The possible role of this passage in protein-losing enteropathy is pointed out.

The usefulness of Ketostix as a semiquantitative screening procedure for the estimation of acetoacetate concentration in both plasma and urine has been established. Because of the reasonably constant relationship of β-hydroxybutyrate to acetoacetate, Ketostix also gives a good indication of the (β-hydroxybutyrate concentration. By reading the Ketostix after thirty seconds instead of the recommended fifteen seconds, the sensitivity of the reaction is increased. The use of Ketostix to examine the plasma from patients with diabetic acidosis will reliably distinguish ketotic from nonketotic cases and is strongly recommended for this purpose.

Paper electrophoretic fractionation in barbiturate (barbital) buffer, pH 9, of iodine-substituted insulin 0–10.8 I/mole showed that substitution with 4–6 I/mole influences the binding of zinc to a demonstrable extent. The effect appears to be due to substitution in the imidazole groups of the histidine residues. Substitution with iodine in the tyrosine residues seems to be without significance, at least at the lower degrees of iodination. The importance of the histidine residues for the binding of zinc is shown by selective destruction of the imidazole groups by photo-oxidation, sensitized by methylene blue. Carbamylation of the N-terminal α-amino groups in the A- and B-chains with KOCN only slightly influences the ability to bind zinc while carbamylation with fluorescein isothiocyanate in the N-terminal of the B-chain brings about a more pronounced reduction in the zinc binding capacity.