Dexamethasone suppression test: development of a method for simultaneous determination of cortisol and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry.

BACKGROUND: Dexamethasone is a synthetic glucocorticoid and is analogous to cortisol. It is used in the low-dose overnight dexamethasone suppression test (LDODST) to diagnose hypercortisolism in patients suspected to be suffering from Cushing's syndrome (CS). Measuring plasma dexamethasone in conjunction with measuring the amount of cortisol following the LDODST may allow clinicians to improve the diagnosis of CS. METHODS: Plasma samples were cleaned up by solid-phase extraction before analysis. Liquid chromatographic separation was carried out under reversed-phase conditions prior to detection by tandem mass spectrometry. The analytes were determined in the presence of deuterated internal standards cortisol-d4 and dexamethasone-d4. RESULTS: Limit of quantitation (LOQ) was 1.89 nmol/L for dexamethasone and <0.02 µmol/L for cortisol. Recoveries of both analytes ranged from 80.2% to 114.4%. Intra- and interassay coefficients of variation were <15%. The concentration of dexamethasone and cortisol was determined in 62 patients after performing LDODST. Dexamethasone concentrations ranged from 3.0 to 21.5 nmol/L (median 7.4 nmol/L) for 57 of these samples. For five patients the concentration was 0.22 µmol/L). CONCLUSIONS: A method for the simultaneous measurement of dexamethasone and cortisol in human plasma by liquid chromatography/tandem mass spectrometry has been developed and validated. The method is suitable for controlling the compliance to the LDODST and for determining the cortisol plasma concentration after the test. The interpretation of LDODSTs was improved by the simultaneous determination of both analytes.

[Acute anaemia in a Vietnamese patient with alpha-thalassaemia and a parvovirus infection]. / Acute anemie bij een Vietnamese patiënt met alpha-thalassemie en een parvovirus-infectie.

A 14-year-old girl of Vietnamese descent with an unremarkable medical history presented with haemodynamic shock due to severe anaemia. This was caused by an aplastic crisis resulting from the combined effects of a Parvovirus infection and HbH disease. The HbH disease was a result of compound heterozygosity for the South East Asia (SEA) deletion and the Constant Spring mutation in the genes coding for alpha-globin chains (HbH/Hb Bart's). The girl had multiple blood transfusions and recovered. Family investigation revealed that, in addition to these 2 mutations in the alpha-globin gene, some family members also carried the 3.7-kb deletion of the alpha-globin gene, a mutation in the beta-globin gene resulting in HbE, and a novel mutation of unknown clinical significance in the beta-globin gene. This case demonstrates that essentially asymptomatic carriership of thalassaemia can have serious consequences when coupled with a concurrent infection.

[Calprotectin: a fecal marker for diagnosis and follow-up in patients with chronic inflammatory bowel disease]. / Calprotectine: een fecale marker voor diagnostiek en follow-up bij patiënten met chronische inflammatoire darmafwijkingen.

Chronic inflammatory bowel disease (IBD) is characterised clinically by periods of well being interspersed by exacerbations of disease activity. Differentiation between IBD and less severe disorders such as irritable bowel syndrome requires invasive and expensive diagnostic procedures. Diagnostic differentiation between active disease, symptoms due to residual constriction of the fibrotic lumen and functional symptoms is a well-known problem. There are not yet any laboratory parameters with sufficient discrimination in terms of sensitivity and specificity. Colonoscopy and histopathological examination remain the gold standards: in Crohn's disease this may be complex due to the variable localisation of the inflammatory process. Abdominal scintigraphic procedures, although informative, are complex and expensive. The recent assessment of faecal calprotectin, a calcium- and zinc-binding anti-inflammatory protein found in neutrophilic granulocytes and monocytes, offers an attractive alternative as an index of intestinal inflammation. We measured this stable marker in random stool samples from 187 patients including healthy volunteers, patients with endoscopically classified active IBD or IBD in remission, and patients with other gastrointestinal disorders. Disease activity was monitored by clinical symptoms, blood tests and endoscopy. Our results confirm previous literature findings that faecal calprotectin is a promising and useful non-invasive tool in the screening of patients presenting with abdominal pain and diarrhoea. Moreover, calprotectin seems helpful in differentiating between active and non-active IBD and possibly also in the monitoring of disease activity.

[Diagnosis of cystic fibrosis; simple genotyping to rule out the disease preferable to starting with the sweat test]. / Diagnostiek van cystische fibrose; liever eenvoudige genotypering om de ziekte uit te sluiten dan starten met de zweettest.

For historical reasons, the sweat test remains the cornerstone in the diagnostic work-up for cystic fibrosis (CF). If CF is suspected, the sweat test (following pilocarpine iontophoresis) is usually performed first to confirm or rule out the diagnosis of CF. Sweat testing, however, is cumbersome to the patient, prone to technical difficulties and unreliable in young children < 4 weeks as well as in adults because of increasing chloride concentrations with age. False-positive and false-negative results do exist. Simple DNA testing with PCR in peripheral blood for a small number of the most common mutations causing CF, notably the delta F508-mutation in the Dutch population, rules out CF with a likelihood of more than 99%. Because of the high negative predictive value of DNA testing, in combination with its speed, reliability and convenience for the patient, starting the diagnostic work-up for CF with DNA testing can be justified in hospitals which possess the laboratory facilities for this type of test.

Association of polymorphism in the cytochrome CYP2D6 and the efficacy and tolerability of simvastatin.

OBJECTIVE: Because clinical data about the therapeutic consequences of polymorphic oxidation of simvastatin by CYP2D6 have not been well reported, we sought to investigate the possible link between polymorphism of CYP2D6 and the efficacy and tolerability of simvastatin treatment in a group of 88 patients with hypercholesterolemia. METHODS: The CYP2D6 genotype was determined with use of polymerase chain reaction and restriction fragment analysis, whereas the CYP2D6 phenotype was determined by monitoring the dextromethorphan metabolism. RESULTS: Four of 5 patients with 2 defective CYP2D6 alleles discontinued the therapy at a low daily dose because of adverse events, with a significant mean decrease in the cholesterol levels of 0.23 mmol/L per milligram of simvastatin in the daily dose. In the group of 28 patients with 1 mutated CYP2D6 gene, 13 did not tolerate the therapy, whereas a mean decrease in the cholesterol levels of 0.20 mmol/L per milligram of simvastatin was found. One patient with a multiplication of the CYP2D6 gene showed a cholesterol reduction of only 0.01 mmol/L per milligram of simvastatin, at a maximal daily dose of 40 mg. Only 9 patients of the group of 54 persons who were homozygous for the wild-type allele discontinued the therapy because of intolerance. In that group, a mean decrease of cholesterol of 0.10 mmol/L per milligram of simvastatin was observed. CONCLUSIONS: Our data provide evidence that the cholesterol-lowering effect of simvastatin is influenced by CYP2D6 polymorphism. The clinical use of this knowledge may allow for more efficient individual therapies.

Beta 3-adrenergic receptor gene polymorphism and type 2 diabetes in a Caucasian population.

AIM: The beta 3-adrenergic receptor (beta 3-AR) is suspected to play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. A mutation in the beta 3-AR gene (Trp64Arg) has been associated with the capacity of weight gain and with early onset of noninsulin dependent diabetes mellitus (type 2 diabetes). In this study we investigated the prevalence of the two beta 3-AR alleles in a Caucasian population and studied the association between the beta 3-AR genotype and metabolic disorders (obesity and type 2 diabetes). METHODS: Genomic DNA extracted from peripheral blood leucocytes of 200 Caucasian subjects (137 subjects with and 63 subjects without type 2 diabetes). The MvaI polymorphism of beta 3-AR, which detects the Trp64Arg mutation, was determined by polymerase chain reaction (PCR). We studied the correlation between the Trp64Arg mutation and the body mass index (b.m.i. kg/m2). RESULTS: There was no significant difference between the patients with type 2 diabetes and control subjects in the frequency of the Arg64 allele (5.5% and 4.8%, respectively). Within the group of type 2 diabetes patients were 14 subjects with the Trp64Arg mutation (b.m.i., mean +/- s.d.: 31 +/- 8.5 kg/m2) and 123 without the mutation (b.m.i. 29 +/- 4.8). There was no association between the beta 3-AR gene polymorphism and sex, obesity, blood pressure, glycohaemoglobin concentration, proteinuria. CONCLUSION: Our results suggest that the Trp64Arg mutation is not a major determinant of metabolic disorders (type 2 diabetes, obesity) and chronic complications of type 2 diabetes in a Dutch population.

Apoptosis inducers endotoxin and Fas-ligation enhance the expression of vascular endothelial growth factor in human endothelial cells.

Endothelial cells in culture were exposed during four hours to the apoptosis inducing agents endotoxin (lipopolysaccharide, LPS) and Fas-ligand mimicking antibody in various concentrations. With addition of a deletion primer as internal standard a competitive RT-PCR was performed to measure semi-quantitatively the expression of mRNA of Vascular endothelial growth factor (VEGF). It appeared that endothelial cells survive increasing amounts of LPS and show a concentration- and time-dependent increase in the expression of VEGF-mRNA. The same effect was found with Fas-ligation, although at high concentrations Fas-ligation induced no further increase, but even a decrease of VEGF expression, possibly related to cell damage. Apoptotic cells were rarely observed after LPS-stimulation, but simultaneous incubation with a blocking antibody to VEGF resulted in a significant increase in apoptosis. We hypothesize that endothelial cells are resistant to apoptosis induction by autocrine expression of VEGF under stress conditions.

Adenylosuccinase deficiency with neonatal onset severe epileptic seizures and sudden death.

We report a male infant with adenylosuccinase deficiency who developed epileptic seizures on the second day of life. Growth was normal and seizures were well controlled with anti-epileptic drugs. Despite axial hypotonia associated with peripheral hypertonicity he presented some development until seven months of age, when he developed high fever and died within a few hours. Although clinical heterogeneity in this disorder of purine synthesis and interconversion is well-known, in 14 out of 17 cases who experienced epilepsy seizures started after the first year of life. The early presentation in our index patient followed by his sudden death at the age of 7 months has not been described before. A search for disorders of purine metabolism should be included in the screening programme for every child with severe neonatal convulsions.

Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27.

To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.

[Type D Sanfilippo disease in an 8-year-old boy; a rare cause of mental retardation]. / De ziekte van Sanfilippo type D bij een 8-jarige jongen; een zeldzame oorzaak van mentale retardatie.

This case report describes the history of an 8-year-old boy of full Dutch extraction with mucopolysaccharidosis type III D (Sanfilippo's syndrome type D). Learning problems started at age 5 years; deterioration is only slow at present, and consistent with most of the cases described in literature. Eight of the nine cases described in the literature so far are known to be of Italian origin. Eye investigations showed bilateral peripheral cataract and a fine diffuse white corneal opacity. Ocular abnormalities of this kind have not been reported before in this type of Sanfilippo's syndrome.

Automated determination of angiotensin-converting enzyme in serum.

This is an adaptation of the Fujirebio "ACEcolor" kit for automated measurement of angiotensin-converting enzyme (EC 3.4.15.1) in serum with the Cobas Fara centrifugal analyzer. The linear range extends to an activity of 110 U/L. Results obtained by the present method and by the manual method were identical, and correlated closely (r = 0.983) with those by Cushman's modified method. The reference interval for 77 adult blood-bank donors was 9-25 U/L (mean 17, SD 4 U/L). Within-run and between-run CVs are 1.7 and 4.0%, respectively. The present method permits rapid, precise, and economical measurement of the enzyme and allows users of a Cobas Fara centrifugal analyzer to introduce a fully automated assay for angiotensin-converting enzyme into their clinical laboratory.

Improved specificity of the PABA test with p-aminosalicylic acid (PAS).

Until now use of the PABA test together with [14C] PABA to calculate the PABA excretion index has probably been the best adaptation suggested to enhance the specificity of this non-invasive pancreatic function test. Drawbacks of the method are the application of radioactivity, the fact that children, pregnant women, and patients with renal insufficiency have to be excluded from the test, and the possible interference of drugs and isotopes. We propose simultaneous administration of p-aminosalicylic acid (PAS) in the PABA test and quantification of the urinary PABA and PAS excretion with liquid chromatography. Urinary PABA and PAS excretion in six hours are comparable (69.5 +/- 8.4% and 65.6 +/- 18.4% respectively in five healthy volunteers). Application of the PABA/PAS ratio was compared with the urinary PABA excretion in 21 normal controls, 38 patients with pancreatic disease, and 42 patients without pancreatic pathology. The PABA/PAS ratio and the per cent PABA excretion correlated very well in pancreatic patients: (PABA/PAS ratio) = 0.0149 (% PABA) + 0.052 (r = 0.902). Use of the PABA/PAS ratio enhanced the specificity of the test from 76 to 89%.

Gel isoelectric focusing and IgG index for demonstration of intrathecal IgG synthesis in neurological disorders.

Cerebrospinal fluid (CSF) and serum were obtained from 37 patients with multiple sclerosis and from 112 patients with various other neurological diseases, and the concentrations of IgG and albumin were quantitated immunochemically. Simultaneously, isoelectric focusing (IF) was carried out for the detection of CSF-specific oligoclonal bands in the alkaline region. CSF-specific components were found in 51 patients by IF, the estimated number of which correlated significantly (Rs = 0.733, p less than 0.0001) with the value of the IgG index. In addition, six patients without CSF-specific abnormalities on IF had an increased IgG index (greater than 0.70). In two of them this increase is, in all probability, false-positive; three others showed fully identical IF bands in both CSF and serum. On the other hand, 13 of the same 51 patients showed a normal IgG index (less than 0.70) although CSF-specific components were detectable on IF. These results confirm the superiority of IF over the immunochemical quantitation of albumin and total IgG for the evaluation of immune reactivity in the central nervous system.