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Background GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis causing developmental delays or intellectual disability in untreated patients as a result of irreversible brain damage occurring prior to diagnosis. Normal neurodevelopmental outcome has been reported in patients treated from neonatal period highlighting the importance of early treatment. Methods Five hundred anonymized newborns from the National Newborn Screening Program of The Netherlands were included into this pilot study. Direct sequencing of the coding region of the GAMT gene was applied following DNA extraction. The disease causing nature of novel missense variants in the GAMT gene was studied by overexpression studies. GAA and creatine was measured in blood dot spots. Results We detected two carriers, one with a known common (c.327G>A) and one with a novel mutation (c.297_309dup (p.Arg105Glyfs*) in the GAMT gene. The estimated incidence of GAMT deficiency was 1:250,000. We also detected five novel missense variants. Overexpression of these variants in GAMT deficient fibroblasts did restore GAMT activity and thus all were considered rare, but not disease causing variants including the c.131G>T (p.Arg44Leu) variant. Interestingly, this variant was predicted to be pathogenic by in silico analysis. The variants were included in the Leiden Open Variation Database (LOVD) database (www.LOVD.nl/GAMT). The average GAA level was 1.14 μmol/L ± 0.45 standard deviations. The average creatine level was 408 μmol/L ± 106. The average GAA/creatine ratio was 2.94 ± 0.136. Conclusion The estimated incidence of GAMT deficiency is 1:250,000 newborns based on our pilot study. The newborn screening for GAMT deficiency should be implemented to identify patients at the asymptomatic stage to achieve normal neurodevelopmental outcome for this treatable neurometabolic disease. Biochemical investigations including GAA, creatine and GAMT enzyme activity measurements are essential to confirm the diagnosis of GAMT deficiency. According to availability, all missense variants can be assessed functionally, as in silico prediction analysis of missense variants is not sufficient to confirm the pathogenicity of missense variants.

Objective To study the performance of the first-trimester combined test between 2004 and 2006 compared to a previous period to investigate changes in time and identify reasons for sub-optimal performance. Methods Serum samples were analysed for pregnancy-associated plasma protein A (PAPP-A) and the free β subunit of human chorionic gonadotrophin (fβ-hCG). Nuchal translucency (NT) was measured between 10 and 14 weeks. Tests were considered screen positive, if their calculated Down syndrome (DS) risk was at least 1 in 250 at term. Results A total of 20 293 singleton pregnancies were included in the analysis. The median maternal age fell from 35.7 to 34.3 years. The overall median weight-corrected multiple of the median (MoM) values of PAPP-A and fβ-hCG were 1.12 and 1.03, respectively. The median MoM value of NT was 0.89 and increased from 0.82 to 0.96. Sixty-six DS cases were detected by the screening test. The detection rate (DR) for DS was 75.9%, with a FPR of 3.3%. Conclusion The performance of the first-trimester test has improved over the years. A better performance of the NT measurement was the main reason, although NT assessment should further be improved. In addition, a better setting of the medians for the biochemical parameters may contribute to a higher DR. Copyright © 2009 John Wiley & Sons, Ltd.

Objectives To determine whether estimation of gestational age (GA) in the context of first-trimester Down syndrome screening is standardized in the Netherlands. Methods This was a retrospective study, carried out between January 2005 and December 2006, of women who underwent first-trimester Down syndrome screening (n = 40 730) based on GA, maternal serum analysis and nuchal translucency (NT) measurement. Date of the last menstrual period (LMP), dating scan information including measurement of crown–rump length (CRL), NT thickness and name of the sonographer were recorded for all pregnancies. The accuracy of estimation of GA was evaluated by comparing the GA based on the LMP with that estimated from the CRL, using relevant subsets of the database. A survey of 104 sonographers was performed to further investigate the findings of the preceding analysis. Results In 44% of all first-trimester combined tests the estimation of GA was based on the dating scan; the method of determination of GA was unknown in 23%. In 15% of all cases a dating scan was recorded but was not used to provide the estimation of GA at blood sampling. Detailed analysis showed that a consistent methodology for the estimation of GA from CRL was not maintained within hospitals and obstetric practices. For a single CRL, the reported GA differed by up to 10 days. Finally, it was demonstrated that individual sonographers reported different GAs for a given CRL. Conclusions Currently, estimation of GA in the first trimester in the Netherlands is not standardized. To improve the performance of prenatal screening for Down syndrome, estimation of GA should be based on ultrasound examination, with one nationally accepted CRL curve. Copyright © 2008 ISUOG. Published by John Wiley & Sons, Ltd.

A recent change in the Dutch governmental policy gave way to the development of a national program for prenatal screening for Down syndrome and neural tube defects (NTD). Here, we present an overview of the current situation on screening for Down syndrome and NTD in the Netherlands. Secondly, we describe the outline of the emerging organisation of such a national programme. Finally, we present one of the current concerns in the Dutch screening practice, namely the fact that several software programmes for risk calculation, producing dissimilar results, are in use. We stress the importance of central direction of the screening programme to overcome this and other problems.

Background: Because of lack of worldwide standardization of influenza virus surveillance, comparison between countries of impact of a pandemic is challenging. For that, other approaches to allow internationally comparative serosurveys are welcome. Objectives: Here we explore the use of neonatal screening dried blood spots to monitor the trends of the 2009 influenza A (H1N1) pdm virus by the use of a protein microarray. Study design: We contacted colleagues from neonatal screening laboratories and asked for their willingness to participate in a study by testing anonymized neonatal screening bloodspots collected during the course of the pandemic. In total, 7749 dried blood spots from 13 countries in 5 continents where analyzed by using a protein microarray containing HA1 recombinant proteins derived from pandemic influenza A (H1N1) 2009 as well as seasonal influenza viruses. Results: Results confirm the early start of the pandemic with extensive circulation in the US and Canada, when circulation of the new virus was limited in other parts of the world. The data collected from sites in Mexico suggested limited circulation of the virus during the early pandemic phase in this country. In contrast and to our surprise, an increase in seroprevalence early in 2009 was noted in the dataset from Argentina, suggestive of much more widespread circulation of the novel virus in this country than in Mexico. Conclusions: We conclude that this uniform serological testing of samples from a highly standardized screening system offers an interesting opportunity for monitoring population level attack rates of widespread diseases outbreaks and pandemics. (C) 2014 The Authors. Published by Elsevier B.V.

This report describes the results of the second part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent report we described the characteristics of LH monoclonal antibodies. In the present work we focused on the comparison of 11 commercially available monoclonal antibody based kits and one polyclonal antibody based RIA. Recovery experiments of the second IS 80/552 and of a related LH preparation showed that curve calibration differed among kits. The ratio between the LH recovery of the two most different kits was 2.30. LH concentrations were determined, using the 12 assay methods in the sera of prepubertal children (n = 46), normal women (n = 26) and men (n = 39) before and after LHRH stimulation, post-menopausal women (n = 29) and patients with renal failure (n = 71) or polycystic ovaries (n = 28). In children LH was detected in 0 to 80% of the sera depending on the kit. In adults, the mean LH concentration provided by the 12 kits varied from 6.0 to 13.55 IU/L showing a ratio of 2.26 between the two most different kits. A thorough statistical analysis allowed to distinguish two groups of kits. The first groups consisting of 6 kits provided results close to those obtained by the RIA. The 5 other kits misrecognized circulating LH in subjects with various clinical status. These 5 kits were the only ones to use, as labelled probes, monoclonal antibodies specific for the holohormone (anti-alpha beta) whereas the 6 other kits used, as labelled probes, monoclonal antibodies directed to the alpha (1 kit) or the beta-subunit (5 kits). In both groups, the coated monoclonal antibodies are directed to either the beta-subunit (1/6 and 2/5 kits) or the holohormone (5/6 and 3/5 kits). Taken together these data suggest that discrepancy among LH assay kits is related to variation in standard curve calibration and in epitope specificity of monoclonal antibodies used in the kits. These findings may prove to be instrumental to alleviate differences among LH assay methods.

Objective This report provides an overview of 15 years prenatal screening for Down syndrome (DS). Methods Between 1991 and 2005, blood samples for the triple test were sent for analysis to our laboratory. Test results were considered screen-positive for neural tube defects (NTDs) if the serum alpha-1-fetoprotein ≥ 2.50 MoM for singleton pregnancies or screen-positive for DS if the calculated risk was at least 1 in 250. Results As many as 42 554 tests were performed. Data on the pregnancy outcome were available for 30 290 screening tests (71.2%). In 1991, most requests (93%) came from the university hospitals; thereafter a shift toward midwives occurred. Until 2001, the number of requests rose to 3500 a year. Most samples were collected between 15 and 17 weeks of gestation. The median age of women for whom a test was requested increased from 30.5 to 34.5. The detection rate (DR) for DS remained stable over the years (80%), with a false positive rate of about 13%. The DR for Trisomy 13, 18, and NTD was 50, 68, and 70%, respectively. Conclusion Based on the results of this study, the triple test may be considered a fairly good second trimester screening test. Here it is shown that health practitioners got more acquainted with the test through the years. This may have served the swift introduction of a formal national screening program that started in January 2007. Copyright © 2008 John Wiley & Sons, Ltd.

The outcome was determined of population-wide neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using tandem mass spectrometry (MS/MS) in The Netherlands, between October 2003 and September 2005. Prospective population-wide neonatal screening for MCAD deficiency was performed in the northern part of The Netherlands. In newborns with blood octanoylcarnitine (C(8:0)) concentrations > or =0.3 micromol/L, clinical and laboratory follow-up was initiated, including MCAD enzymatic measurements which played a decisive role. In a 2-year period, 66 216 newborns were investigated for MCAD deficiency and follow-up was initiated in 28 newborns. True-positives (n = 14) were identified based upon MCAD enzyme activity 5.0 turned out to differentiate between false-positives and true-positives. Measurement of MCAD activity using phenylpropionyl-CoA as a substrate further discriminated between newborns with MCAD deficiency and so-called mild MCAD deficiency. To summarize, neonatal screening for MCAD deficiency in the northern part of The Netherlands resulted in the predicted number of affected newborns. Measurement of MCAD activity in leukocytes or lymphocytes using phenylpropionyl-CoA as a substrate can be regarded as the gold standard to diagnose MCAD deficiency upon initial positive screening test results

OBJECTIVES: This is the first report on the results of a first-trimester combined-test screening programme in the Netherlands in a multi-centre routine clinical setting. METHODS: Between July 2002 and May 2004, blood samples were taken from subjects in 44 centres in the Netherlands and sent to our laboratory to assay for maternal serum concentrations of fbeta-hCG and PAPP-A. Fetal nuchal translucency (NT) was measured in the participating centres at a gestational age (GA) of 10-14 weeks. Results of those pregnancies for which a combined biochemical and NT risk was calculated were included in the epidemiological analysis of this study. RESULTS: A total of 4033 singleton pregnancies were included in the analysis. The median maternal age of the analysed group was 36.5 years. The distribution of GA was biphasic, with median GA of 10.3 and 12.1 weeks, respectively. The detection rate using the combined ultrasound and serum screening at a cut-off level of 1 in 250 was 71% (15/21), with a screen-positive rate of 4.7%. CONCLUSION: The results of this study show that the first-trimester combined test is suitable as a prenatal screening test in a multi-centre routine clinical setting in the Netherlands. Strict performance evaluation should identify weaknesses in the organisation that impair the performance of the test. Here, the performance of NT was especially identified as a candidate for improvement.

Objective To develop a standard preparation for measurement of phenylalanine in blood spots in phenylketonuria against which manufacturers of kits for phenylalanine determination could calibrate their own blood spot calibrators. Methods As most of the variation of phenylalanine concentration in dried blood spots is due to the way the blood spot calibrators are prepared, a minimum set of prerequisites for the preparation of blood spot calibrators was established by a group of scientific experts and manufacturers. A “European working standard for phenylalanine in blood spots” (EWS-Phe-01) was prepared and distributed to manufacturers taking part in this project. Results The control of the homogeneity, linearity, and stability of the EWS-Phe-01 batch gave results within expected ranges. The preliminary results of the recalibration of calibrators for phenylalanine against EWS-Phe-01 by the participating manufacturers were promising; the differences between measurements were much decreased. Conclusion Manufacturers have agreed to use EWS-Phe-01, and since November 1996 EWS-Phe calibrators have been routinely available from these companies. This pilot study may act as a model for minimising discrepancies found in other screening tests.

Incubation media of pituitary cells and homogenates of pituitary tumors were studied by isoelectric focusing followed by radioimmunoassay. Almost all LH-immunoreactive components detected had been observed in earlier studies on highly purified preparations. In one pituitary tumor homogenate, intact LH (LHi) was detected. The majority of LHi-components present in this tumor were relatively acidic (LH Type I) components. The corresponding beta-subunit population was in agreement with the one earlier observed for Type I LH. In all other tumor homogenates only alpha- and beta-subunits could be detected. In incubation media of pituitary cells from patients with no known endocrine disease nor a pituitary tumor highly acidic populations of alpha-subunits were detected. In only one of the incubation media significant amounts of LHi and free beta-subunits were observed. In an incubation medium of a TSH-producing pituitary tumor significant amounts of intact LH, free alpha- and beta-subunits were detected. The cell contents of this tumor contained components with lower pl-values than the medium. In general a good correlation is observed between the population of LH-components in material from individual identifiable pituitary glands and in highly purified preparations from large numbers of anonymous pituitary glands.

To evaluate the effectiveness of the national programme for prevention of perinatal hepatitis B infections.The regional public health laboratories and provincial immunization administrations in the Netherlands.Retrospective evaluation.Starting October 1989 routine screening of pregnant women for HBsAg was performed and passive-active immunisation of infants of HBsAg-positive mothers was added to the national immunisation programme. Infants receive hepatitis B immunoglobulin at birth and hepatitis B vaccine at 3,4,5, and 11 months of age, concomitant with the DTP-polio vaccine. The effectiveness of screening and intervention in 1990 was evaluated.Screening covered about 85% of the pregnant population and the prevalence (0.44%) was less than expected. About 60% of the infants born to HBsAg-positive mothers were registered for vaccination. Of these infants the average coverage was 83% for immunoglobulin, and 90%, 86%, 80% and 55% for the four successive hepatitis B vaccinations. There was considerable delay in vaccine administration; frequently doses were administered later than recommended.Compliance with screening and vaccination appeared incomplete. Recommendations for the simplification of the current programme are made.

Evaluation of the Dutch screening programme for congenital hypothyroidism (CH).Descriptive.Nationwide.Data on the screening were obtained from the administration body for vaccination and screening results, laboratories and paediatricians to whom infants with positive screening values were referred, during the period from January 1st, 1981 to December 31st, 1990.Of all live births in the Netherlands, 99.5% (1,797,719) were screened for CH. During the study period, 10,165 children (0.57% of all screened infants) were referred. Of the referred children, 529 had primary CH and 53 had congenital thyrotropin deficiency syndrome (CTDS). The prevalences of primary CH and CTDS are 1:3,400 and 1:25,000, respectively. The sensitivity of the programme with respect to detection of primary CH and CTDS was 99% and 74%, respectively. For all forms of CH combined, the specificity of the programme was 99% and the positive predictive value 6%. The positive predictive value was especially low in the group of infants with low T4 and normal thyrotropin values. One of the goals of the programme is to realise the start of treatment in all patients before they reach the age of three weeks. Before the screening programme came into being, the cumulative proportion of patients treated on the 21st day was 6%. After the beginning of the programme, the proportion increased to 54%. In screened patients with a severe form of CH it is currently 72%.The screening programme has made a substantial and important contribution to early and effective identification of patients with CH. A number of measures to decrease the number of false-positive results have been taken and others are at present being investigated. Although patients are now treated much earlier than before the programme started, substantial improvement in this respect is still possible. This can only be achieved by a collective effort of performers of the heel puncture, laboratories, administration body for vaccination and screening results, general practitioners and pediatricians.

Spontaneous morphine withdrawal has been studied in rats after induction of dependence using administration of morphine via the food (+/- 160 mg/kg/day on the basis of body weight). In the present paper, the decrease of the nocturnal locomotor activity and of food intake were validated as real morphine withdrawal symptoms. During the withdrawal phase, morphine was readministered either by subcutaneous injection of morphine or admixed in the food. Subcutaneous administration resulted in short-lasting enhancements (of 50%) of the locomotor activity (peak effects) followed by a decrease of 50%, while food intake and loss of body weight were hardly affected. Administration via the food resulted in a dose-dependent reduction of the decrease of the nocturnal locomotor activity, the decrease in food intake and the loss of body weight. Chronic naloxone infusion (0.03 or 0.1 mg/kg/hr) via osmotic minipumps appeared to have similar effects on locomotor activity, food intake and body weight, compared with spontaneous withdrawal of morphine. However, the recovery to normal values appeared to occur faster after naloxone infusion. It is concluded that the observed withdrawal symptoms, i.e. decrease of the locomotor activity, decrease of food intake and loss of body weight, can be interpreted as real withdrawal symptoms not caused by additional manipulations occurring in the course of the experiments.

We studied the effect of Cyclosporin A on the thymus in rats. A daily subcutaneous injection of Cyclosporin A at a dose of 45 mg/kg body weight in young adult rats resulted in a 100% mortality within two weeks. Daily administration of 15 mg/kg for two weeks was well tolerated. Rats treated at this dose showed whole blood Cyclosporin concentrations around 6 mg/l. The thymus immediately after treatment showed the almost complete disappearance of the medulla, including the microenvironment made by medulla-type epithelium, interdigitating dendritic cells defined by their expression of MHC class II antigen, and lymphocytes with a mature T-cell phenotype. After discontinuation of Cyclosporin treatment, a rapid recovery occurred, with the reappearance of medulla areas after 2 weeks. These areas differed from the normal medulla by the absence of medulla-type epithelium. This cell population recovered in its normal location in about 4 weeks. The reappearance of medullary interdigitating cells was associated with reappearance of lymphocytes with a mature T-cell phenotype. In the regenerating thymus "holes" in the microenvironment were observed that lacked epithelium and interdigitating cells, and that were filled by lymphocytes with an immature cortex phenotype. Peripheral lymphoid organs, of which the spleen was studied in more detail, did not manifest changes in lymphoid and stromal components. Target organs for syngeneic graft-versus-host disease, as described under special conditions after Cyclosporin treatment, did not show any histologic abnormality. The changes in thymus (immuno)histology may be associated with changes in shaping the T-cell repertoire: but, clinical manifestations of such changes require special experimental conditions.

Summary. In a series of experiments with polycythaemic mice the influence of liver enzyme inhibitors on erythropoietin activity was studied. SKF 525-A was found to increase the half disappearance time of the activity of administered sheep erythropoietin. Both the administration of SKF-525-A and Disulfiram were found to increase the half disappearance time of endogenous erythropoietin activity. It is concluded that these compounds block the inactivation of erythropoietin in the liver. A model in which the action of such inhibitors on the disappearance of endogenous erythropoietin activity can be tested, is proposed.

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.

A highly purified LH preparation, prepared from human pituitaries (NM14) was studied using immunochemical and in vitro biological techniques. Using isoelectric focusing 5 different biologically active components could be detected, 4 of which were located between pH = 7.0 and 8.6, one was located at pH = 4.9. The biological activity in the acidic part of the pH gradient is probably due to the formation of an artefact during storage in solution. The experiments were performed with special emphasis on the occurrence of LH subunits, for which until now no pI-values have been reported. Using specific radioimmunochemical (RIA) systems at least 7 different α-subunit components and 4 different β-subunit components could be detected. The presence of even more components is likely. The α-subunit components, with pI-values of: 4.6, 5.2, 6.0, 7.1, 8.1, 8.8 and 9.7, were located spread over the entire pH-gradient whereas all β-subunit components were located above pH = 7.5, the pI-values being 7.7, 8.4, 8.5 and 10.4. The identification of these components as α- or β-subunit was based on the relative response in the different RIA systems, the absence of biological activity and the response changes during incubation at 37°C. Refocusing of the above mentioned biologically active components individually resulted each time in a single component with a pI-value identical to its corresponding 'parent'. After incubation at 37°C of these components each time the same variety of subunit components was found with discrete pI-values, identical to those above.

The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free α-or β-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free α- or β-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the α-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the β-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM 14) which was prepared by one of us and on which we have reported earlier. In addition, with the abovementioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37°C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, α- and β-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.

The uterotropic effect of beta-HCH was assessed in juvenile female mice and rats. A dose of 50 mg/kg feed over five days gave a significant increase in the uterine weight. The potency of beta-HCH compared to 17 alpha-ethynyloestradiol was about 20 X 10(-6). Based on these and other as yet unpublished observations, it is concluded that beta-HCH has oestrogenic features.

Pertussis vaccine consisting of inactivated whole Bordetella pertussis organisms appears to induce a biphasic response on the glucose metabolism of N:NIH mice. A heat stable component assumed to be the LPS induces a transient hyperglycaemia within 6 hours after vaccination. A heat labile component assumed to be LPF, induces a hypoglycaemia and hyperinsulinaemia 2-7 days after treatment. A purified vaccine examined in this study still showed some effects on the glucose metabolism at 3-4 days after vaccination. If hypoglycaemia contributes to the neurological side effects, incidentally observed after vaccination of infants, both LPS and LPF have to be considered to be responsible for these effects.

Male and female Wistar rats were fed bis(tri-n-butyltin)oxide (TBTO) at 0, 5, 20, 80, or 320 mg/kg diet for 4 weeks. Clinical signs and decreases in feed and water consumption were observed in the 80 and 320 mg/kg groups. The serum transferase activities (alanine amino transferase and aspartate amino transferase were increased at 20 (males only), 80, and 320 mg/kg. The serum glucose and liver glycogen concentrations were lowered in the 320 mg/kg group. At 80 and 320 mg/kg the serum IgG level was reduced and IgM level was increased. Compared to controls the mean relative weight of the thymus was decreased at 20 (males), 80, and 320 mg/kg. In the groups receiving 80 or 320 mg/kg microcytic anemia was found. The white blood cell counts were decreased, due to the reduction in the number of lymphocytes in the 80 (males) and 320 mg/kg groups. The concentration of neutrophilic granulocytes was increased in the highest dose group. Histopathologic effects included a dose-related lymphocyte depletion of thymic cortex and of T lymphocytes in spleen and mesenteric lymph nodes. In the spleen also depletion of iron stores was found, and in the medullary sinuses of mesenteric lymph nodes, rosettes of erythrocytes were found around mononuclear cells; the occurrence of rosettes increased with dose from 5 to 80 mg/kg, and appeared to be the most sensitive parameter. A low incidence of areas of liver necrosis with inflammatory reaction and bile duct hyperplasia was found in the 320 mg/kg group. A viral or bacterial etiology could be demonstrated for these liver lesions, but they appeared associated with TBTO-induced ulcerative inflammation of the common bile duct as shown in an additional study. In 6-week studies exposure of male weanlings to the 0, 20, and 80 mg/kg diets, the serum insulin concentration in the treated groups was decreased, although the response to glucose challenge was unaffected. The serum thyroxin and thyrotropin (TSH) concentrations were reduced, whereas the luteinizing hormone (LH) concentration was increased in the 80 mg/kg group. The concentrations of follicle-stimulating hormone (FSH) and corticosterone were not changed. The release of LH and FSH was enhanced in the 80 mg/kg group and a tendency toward reduced release was found for TSH. Using immunocytochemistry a dose-related reduction was found in the number and staining intensity of TSH-producing cells in the pituitary, correlating with histopathologically decreased activity of the thyroid.(ABSTRACT TRUNCATED AT 400 WORDS)

The charge heterogeneity of an LH preparation containing relatively acidic components (LH Type I) was studied. Four biological active components, with pI-values of 5.04, 5.60, 6.06 and 6.57 were detected. A total of four different α-subunits, with pI-values of 4.49, 4.79, 5.16 and 6.02 could be detected after incubation at 37°C. With the exception of the most acidic component all these α-subunits were also present in earlier studied LH Type II preparations. After neuraminidase treatment a strong shift to more basic components was observed, resulting in a population of components similar to the one detected in LH Type II preparations. The β-subunits detected were very different from those observed in Type II preparations. All six components detected had pI-values < 7.5. Upon incubation at 56°C these subunits appeared to be unstable resulting in a shift to more basic pI-values, these pI-values being very similar to those of β-subunits observed before in Type II preparations. After neuraminidase treatment, the pH values of the population of β-subunits became identical to those of the population in LH Type II. From these results it is concluded that the major charge difference between LH Type I and Type II is located in the β-subunits. This difference cannot be explained completely by differences in sialic acid content, but may also be due to heat labile charged groups such as sulphate.

Summary From the results of this study the conclusion can be drawn that radioimmunoassay (RIA) of diethylstilbestrol (DES) in crude urinary extracts may lead to highly unreliable results. Simple paper‐chromatographic purification, prior to radioimmunoassay, eliminates interfering substances responsible for ‘false DES’ values. Such a purification step is mandatory, certainly when the radioimmunoassay is used for statutory control purposes.

beta-Endorphin (beta-LPH61-91) is a well known endogenous opioid ligand. It and related peptides have recently been implicated in the control of adaptive behaviour. Smaller beta-endorphin fragments appeared to be more active moieties than the parent molecule in a number of behavioural situations. Their effects seemed to occur independently of interaction with opiate receptor sites in the brain. Moreover, elimination of the opiate-like properties of gamma-endorphin (beta-LPH61-77) by removing the N-terminal amino acid yielded des-tyrosine-gamma-endorphin (beta-LPH62-77, dT gamma E) which had greater behavioural activity than gamma-endorphin. The CNS effects of dT gamma E resembled those of neuroleptic drugs in several test systems. alpha-Endorphin (beta-LPH61-76) exerted effects opposite to those of dT gamma E and in some aspects its activity was comparable to that of psychostimulant drugs. This opposition of effects suggests that a balance between gamma- and alpha-type endorphins is involved in the control of brain function. We report here that either gamma-endorphin and dT gamma E or alpha-endorphin and des-tyrosine-alpha-endorphin (beta-LPH62-76, DT alpha E) can be formed preferentially from beta-endorphin by enzymes associated with an enriched synaptosomal plasma membrane fraction from brain. It is suggested that these enzymes have a role in brain homeostatic mechanisms by regulating the generation of these substances.

By preparative isoelectric focussing of a highly purified LH preparation in a sucrose density gradient, four biologically active LH components were isolated. The effect of neuraminidase treatment of each component on the charge heterogeneity was studied by isoelectric focussing followed by in vitro biological and immunochemical techniques. The number of biologically active components with pI-values greater than 7 containing varying amounts of sialic acid is at least six. The pI-value of the most basic (= asialo) LH component was 9.26. The two most basic components were not present in our preparation (NM14) before neuraminidase treatment. It is concluded that the difference between the pI-values of LH components is caused by a difference in sialic acid content. When an intact LH component was incubated with neuraminidase there was detectable dissociation owing to the elevated temperature (37 degrees C) and necessary acidic conditions of the incubation. Under these conditions we found the same subunits as we have described before. The most basic alpha-subunit had a pI-value of 9.29, whereas two beta-subunits with pI greater than 9 were observed at pI 9.26 and pI 9.9. On the other hand, when an LH component was forced to dissociate by incubation at 56 degrees C prior to neuraminidase digestion, two additional alpha-subunits were found. From this it is concluded that in the intact LH molecule, some sialic acid residues are poorer substrates for neuraminidase action than in the free subunits.

Summary: Six commercially available radioimmunoassay (RIA) kits for human follitropin (follicle-stimulating hormone; hFSH) were compared with respect to precision, "accuracy", specificity and performance. The intra- and interassay coefficients of Variation ranged from 4.7 to 8.3% and from 6.5 to 13.3%, respectively. The recovery of added follitropin ranged from 88 to 176%. The most important conclusion of this study is that an accurate calibration of the kit Standard by the manufacturer is essential for minimizing the Variation between kits. « Vergleich von sechs Radioimmunoassay-Bestecks zur Bestimmung von menschlichem Follitropin im Serum Zusammenfassung: Sechs kommerziell erhaltliche Radioimmunassay-Bestecks zur Bestimmung menschlichen Follitropin wurden hinsichtlich Prazision, Richtigkeit, Spezifitat und Durchfuhrung verglichen. Die Variationskoeffizienten betrugen in der Serie 4,7 bis 8,3%, von Tag zu Tag 6,5 bis 13,3%. Die Wiederfindung zugesetzten Foljitropins reichte von 88 bis 176%. Die wichtigste Schlusfolgerung aus dieser Untersuchung ist, das eine richtige Kalibrierung des Standards im Testbesteck durch den Hersteller fur die Verminderung der Variation der mit den Bestecks erzielten Ergebnisse von ausschlaggebender Bedeutung ist.

The characteristics and dynamics of hormone secretion in vivo and in vitro were investigated in six patients with gonadotropin-secreting pituitary adenomas. All six tumors secreted and contained FSH and different combinations of LH, beta-LH, and alpha-subunit. In addition, immunohistochemical examination of the pituitary tumor tissue showed staining with both LH and FSH in three and either LH or FSH in the other three tumors. TRH and GnRH stimulated hormone secretion in vivo and in vitro, and they also increased the hormone content of the cultured tumor cells. Bromocriptine significantly inhibited hormone release and reduced the hormone content of the tumor cells. In vivo, 2.5 mg bromocriptine significantly suppressed plasma hormone levels; the inhibiting effect on alpha-subunit concentrations was in general more marked than that on LH and FSH. We conclude that hormone release by gonadotropin-secreting pituitary adenomas can be stimulated by TRH and GnRH and inhibited by bromocriptine. Most of these tumors synthesize FSH, but there is a wide variation in the production of LH, beta-LH, and alpha-subunits. The sensitivity of hormone release to bromocriptine suggests that chronic therapy with this drug might have a beneficial effect on pituitary tumor size.

The relation of dietary factors and physical activity to hyperinsulinemia was examined in 389 men aged 70-89 years who participated in the Zutphen Elderly Study in 1990. Information about the usual diet was obtained using a cross-check dietary history, and habitual physical activity was assessed using a validated questionnaire. Known and newly diagnosed diabetic patients were excluded from this study, since serum insulin and C-peptide levels are indicators of insulin resistance and hyperinsulinemia in non-diabetics only. Insulin levels during the oral glucose tolerance test were lowest in men with the highest physical activity. This inverse association was independent of age, body mass index, the ratio of subscapular to triceps skinfold thickness, cigarette smoking, and energy intake (p < 0.001). In addition, insulin levels were inversely associated with the intake of dietary fiber and polyunsaturated fatty acids, which could not be accounted for by variables such as energy intake, body mass index, physical activity, prescribed diets, or the presence of coronary heart disease. In contrast, insulin levels increased with the increasing intake of saturated fatty acids and alcohol. The fasting C-peptide level was independently associated with the intake of total fat, saturated and monounsaturated fatty acids, and alcohol, whereas an inverse relation with the intake of total carbohydrates and dietary fiber was seen. Besides overweight, physical activity and dietary factors such as the intake of fatty acids, fiber, carbohydrates, and alcohol, were independently associated with hyperinsulinemia and insulin resistance. Therefore, these behavioral factors may partly determine the occurrence of non-insulin-dependent diabetes mellitus and coronary heart disease and play a role in the prevention of these disorders.

Door het toevoegen van kleurstoffen aan reagentia van RIA systemen is controle op het pipetteren en eventueel morsen (van radioactiviteit) eenvoudig. Een twaalftal in de voedingsmiddelen industrie veel gebruikte kleurstoffen werd in acht in eigen beheer ontwikkelde RIA systemen getest met betrekking tot verstoring van initiele en aspecifieke binding. Vrijwel alle kleurstoffen zijn bruikbaar in een concentratie van 50 mg/l incubatievolume.

Neonatal screening for congenital disorders like phenylketonuria (PKU), congenital hypothyroidism (CH) and congenital adrenal hyperplasia (CAH) is generally performed in dried blood spots on filter paper. The analytes of interest for testing for PKU, CH and CAH are phenylalanine, thyrotropin (TSH) and 17alpha-hydroxyprogesterone (17OHP), respectively. The International Society for Neonatal Screening (ISNS) decided to prepare a combined reference preparation for the three analytes on filter paper SchleicherSchuell #903, Whatman BFC180 and Toyo Roshi 545. This 'First ISNS Reference Preparation for Neonatal Screening for TSH, phenylalanine and 17OHP in blood spots' (1st ISNS-RPNS) has been prepared by the RIVM (Bilthoven).The number of filter paper cards prepared, each with two sets of six blood spot calibrators, was 480, 42 and 69 for SchleicherSchuell #903, Whatman BFC180 and Toyo Roshi 545, respectively. The volume of blood dispensed was 50 microl. The range of concentrations for TSH was 1-121 mIU/L blood, for phenylalanine 65-865 micromol/L blood and for 17OHP 2.2-302 nmol/L blood.The linearity of the blood spot calibrators and the homogeneity of the batch (only tested for SchleicherSchuell) were good. The differences between the three filter papers were small: i.e. the potency of the ISNS-RPNS on Whatman and Toyo Roshi in terms of SchleicherSchuell was between 0.98 and 1.09 for the three analytes.The 1st ISNS-RPNS for TSH, phenylalanine and 17OHP can be said to be suitable as formal reference preparation and as a source for (re)calibrating kit calibrators.