Excretion kinetics of 1,3-dichlorobenzene and its urinary metabolites after controlled airborne exposure in human volunteers.

The solvent 1,3-dichlorobenzene (1,3-DCB) is formed during thermal decomposition of the initiator 2,4-dichlorobenzoylperoxide in the production of silicone rubber with potential exposure of production workers as shown in previous works. Despite a threshold limit value (MAK value) of 2 ppm in air, there are currently no data about the corresponding internal exposure that would allow for the derivation of a biological limit value. In the present study, we have investigated the absorption of 1,3-DCB and urinary kinetics of its metabolites in 10 human volunteers after controlled inhalative exposure. Due to the strong odour of 1,3-DCB, a subjective evaluation of odour nuisance was also performed. Ten male human volunteers (23-36 yrs.) were exposed 6 h/day to a concentration of 0.7 ppm and 1.5 ppm in the Aachen workplace simulation laboratory (AWSL) with one week between each experiment. In order to investigate potential dermal absorption, the volunteers were exposed to 1.5 ppm wearing a suitable filter mask that prevented inhalative exposure in a third exposure. 1,3-DCB in blood was measured after 3 and 6 h exposure and the urinary metabolites 3,5-dichlorocatechol (3,5-DCC), 2,4-dichlorophenol (2,4-DCP) and 3,5-dichlorophenol (3,5-DCP) were measured over 24 h after exposure via LC/MS/MS. There were clear dose-response relations for all investigated parameters. The maximum excretion of the metabolites was reached at the end of exposure and corresponded to 5.2 ± 0.7 mg/g crea, 1.5 ± 0.35 mg/g crea and 0.07 ± 0.011 mg/g crea at 0.7 ppm and to 12.0 ± 3 mg/g crea, 3.5 ± 1.1 mg/g crea and 0.17 ± 0.05 mg/g crea at 1.5 ppm for 3,5-DCC, 2,4-DCP and 3,5-DCP, respectively. The use of filter masks decreased the internal exposure for about 85-90%, indicating substantial dermal absorption. Odour perception did not show a dose-response, probably due to fast olfactory adaption. The human study presented here provides an excellent basis for deriving a biological limit value for 1,3-DCB.

[Don't be guided purely by numbers: false increased TSH values due to analytical interference]. / Vaar niet alleen op een getal.

BACKGROUND: Physicians are often guided by laboratory values. When a clinical presentation does not match laboratory values, one must consider the possibility that these values may be falsely increased or decreased. A common cause is analytical interference. CASE DESCRIPTION: A 57-year-old male, presenting with fatigue and palpitations, had high TSH and normal FT4 values. Although there were no fitting clinical symptoms for these values, the patient was treated with levothyroxine assuming he had subclinical hypothyroidism. TSH levels remained high, however, whereas FT4 levels increased and the patient developed thyrotoxicosis. Eventually, it was discovered that the TSH was falsely elevated. CONCLUSION: The patient turned out to have macro TSH, where TSH forms conjunctions with IgG into larger molecules. These conjugates cause a rarely occurring interference during laboratory analysis, resulting in a falsely increased TSH value.

Online magnetic bead dynamic protein-affinity selection coupled to LC-MS for the screening of pharmacologically active compounds.

The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

Characterization of high molecular weight plasma protein complexes induced by clotting factor rFXIII-treatment in the cynomolgus monkey.

BACKGROUND: In cynomolgus monkeys, suprapharmacological doses of clotting recombinant factor XIII (rFXIII) cause a generalized coagulopathy, associated with formation of circulating high molecular weight protein complexes (HMEX). HMEX consist of plasma protein substrates cross-linked by FXIII transglutaminase activity. OBJECTIVE: To characterize HMEX, with a view to develop safety biomarker assays. METHODS: Cynomolgus monkeys received single i.v. injections with vehicle or rFXIII at 1, 3 and 10 mg kg(-1). Plasma HMEX were analyzed by sodium dodecylsulfate-polyacrylmide gel electrophoresis, silver staining, Western blotting and quantitative dissociation-enhanced lanthanide fluoroimmunoassay. Plasma FXIII antigen was analyzed by quantitative ELISA. Human HMEX were made in vitro, by spiking plasma with thrombin-activated rFXIII. RESULTS: Maximal circulating HMEX levels were reached within 1 h of rFXIII treatment, and remained stable over 24 h. HMEX above 250 kDa contained fibrinogen alpha-chains and fibronectin. Fibrinogen gamma-chain was detected only in HMEX below 250 kDa. The total plasma concentration of HMEX was in the low microg mL(-1) range, distributed on less than 20 main species. Human and cynomolgus HMEX were similar. HMEX formation increased with rFXIII dose in a disproportionate manner, with 3-fold and fortyfold increases in HMEX exposure associated with rFXIII dose increments from 1 to 3 and 3 to 10 mg kg(-1), respectively. CONCLUSIONS: The disproportionate HMEX formation parallels the steep toxicity dose response previously reported for rFXIII in cynomolgus monkeys, supporting a mechanistical role for HMEX in the generalized coagulopathy seen in rFXIII toxicity. Our findings support that HMEX constitute candidate (potential) safety biomarkers in rFXIII treatment.

Preclinical toxicity biomarkers for combination treatment with clotting factors rFXIII and rFVIIa.

Combination treatment with the clotting factors recombinant activated factor VII (rFVIIa), serine protease, and recombinant factor XIII (rFXIII), protransglutaminase, is being explored for haemostatic therapy. We performed a single-dose toxicology study in the cynomolgus monkey, with four dose groups receiving 0.1 + 0.34 mg kg(-1) (group 1), 0.33 + 1.12 mg kg(-1) (group 2), 1.67 + 5.60 mg kg(-1) (group 3) and 5.00 + 16.80 mg kg(-1) (group 4) of a rFVIIa + rFXIII combination. In the three lower dose groups, no clinical, histopathological or blood chemistry changes were observed. In group 4, the animals died at 4 h post-dosing, with histopathology revealing a systemic coagulopathy resembling, but distinct from, disseminated intravascular coagulation. Due to the absence of toxicity warning signs, toxicity biomarkers were identified by a Western blot-based screening of approximately 20 plasma proteins known to be involved in the clotting cascade. Three of the examined proteins were specifically affected by rFVIIa + rFXIII treatment. Fibronectin and fibrinogen exhibited dose-dependent reductions from less than 10% reduction (group 2) to more than 90% reduction (group 4). These reductions were reversible, and specific. For vitronectin, a dose-dependent conversion to the 65-kDa form was found to occur in groups 3 and 4. Thus, fibrinogen, fibronectin and vitronectin represent the first biomarkers for clotting factor toxicity.

Metal-complex formation in continuous-flow ligand-exchange reactors studied by electrospray mass spectrometry.

Electrospray ionization mass spectrometry was used to investigate complex formation of different metal complexes in a continuous-flow ligand-exchange reactor. A computer program was developed based on normal equilibrium calculations to predict the formation of various metal-ligand complexes. Corresponding to these calculations, infusion electrospray mass spectrometric experiments were performed to investigate the actual complex formation in solution. The data clearly show good correlation between the theoretically calculated formation of metal-ligand complexes and the experimental mass spectrometric data. Moreover, the approach demonstrates that the influence of the pH can be investigated using a similar approach. Indirectly, these infusion experiments provide information on relative binding constants of different ligands towards a metal-ion. To demonstrate this, a continuous-flow ligand-exchange detection system with mass spectrometric detection was developed. Injection of ligands, with different affinity for the metal-ion, into the reactor shows good correlation between binding constants and the response in the ligand-exchange detection system. Additional information on the introduced ligand, and the complexes formed after introduction of the ligand, can be obtained from interpretation of the mass spectra.

Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry.

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.

Screening for metal ligands by liquid chromatography--ligand-exchange--electrospray mass spectrometry.

Electrospray ionization mass spectrometry is applied for the selective detection of metal ligands after a post-column continuous-flow ligand-exchange reaction. The detection is based on the specific release of a reporter ligand from a metal-reporter ligand complex by a high affinity ligand. Constant infusion and direct-injection experiments are performed to optimize the method. The on-line coupling of a liquid chromatographic separation prior to the continuous flow ligand-exchange reaction enables the screening for high affinity ligands in complex samples. The feasibility of the method is demonstrated by using several ligands with a different affinity for either Cu(II) or Zn(II) ions. The selectivity of the ligand-exchange detection method can be tuned by the choice of the reporter ligand. This is demonstrated by using either 2,2'-bipyridyl or 5-methyl-1,10-phenanthroline as reporter ligands.

Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.

Accuracy of normal-dose contrast-enhanced MR angiography in assessing renal artery stenosis and accessory renal arteries.

OBJECTIVE: The purpose of this study was to evaluate the accuracy of breath-hold contrast-enhanced MR angiography in the assessment of renal artery stenosis and accessory renal arteries using a standard dose of gadolinium. SUBJECTS AND METHODS: Thirty-eight patients suspected of having renal artery stenosis underwent MR angiography and intraarterial digital subtraction angiography, which was the method of reference. Three-dimensional gradient-echo MR subtraction angiography (TR/TE, 5.8/1.8 msec) was performed on a 1.5-T imager using a phased array body coil. Before imaging, a separate timing bolus sequence was used, administering 1.0 ml of contrast agent. Gadopentetate dimeglumine (15 ml) was injected using an MR power injector. Two observers, who were unaware of each other's interpretation and of MR findings, assessed digital subtraction angiography. Likewise, two other observers assessed MR angiography. RESULTS: Digital subtraction angiography depicted 75 main and 17 accessory renal arteries (n = 92). All main renal arteries and 13 accessory renal arteries were identified on MR angiography. Compared with digital subtraction angiography, MR imaging correctly classified 57 of 66 arteries without a hemodynamically significant stenosis (0-49%), 22 of 22 arteries as significantly stenotic (50-99%), and four of four occluded arteries; five stenoses were overestimated. There was one false-positive finding of an accessory renal artery on MR angiography that was identified retrospectively on digital subtraction angiography. Interobserver agreement was high. Sensitivity and specificity for grading significant stenosis were 100% and 85%, respectively. CONCLUSION: Contrast-enhanced MR angiography, using +/-0.1 mmol/kg of gadolinium, is an accurate method in the assessment of renal artery stenosis and accessory renal arteries.

The application of the International Statistical Classification of Diseases to neurology: ICD-10 NA.

Rapid advances in the clinical neurosciences in the last decade have led to considerable amplification of our ability to classify neurological diseases. For these classifications to be widely used, they must be compatible with the 'International Statistical Classification of Diseases and related health problems' (ICD) of the World Health Organization (WHO), which system is used throughout the world for classification of diseases and reasons of death. The 'Ninth' revision of the ICD (ICD-9), published in 1976, is currently in use in a number of Member States of the World Health Organization, including the United States. However, it is expected that by the end of this decade virtually all Member States will have introduced the 10th Revision of the ICD (ICD-10), published in 1992. An 'Application of ICD-10 to neurology' (ICD-10 NA) has been developed, with a specific coding system for virtually every neurological disease currently recognized. This article describes the structure and background of this work, that is offered as a definitive international classification of neurological disease to be used by clinical and research organizations, governmental and nongovernmental bodies, and for epidemiological and research purposes. It is concordant with the new and proposed classifications of subspecialty neurological organizations.

[The international classification of WHO diseases (ICD-10) and its application in neurology (ICD-10 NA)]. / La classification internationale des maladies de l'OMS (CIM-10) et son application à la neurologie (CIM-10 AN).

The 10th Revision of the International Disease Classification (ICD-10) was published in English in September 1992 and in French in January 1993. Chapter VI (main code G) deals with diseases of the nervous system. There are 11 chapters covering the main categories of aetiologies. The subdivisions designated by the three-character codes are used to define the main diagnostic categories as divided according to aetiology, localization or symptoms. The three-character codes so formed are the legal basis for codifying causes of death in all the member states of the WHO. Supplementary 4-digit codes can be used for more precise definitions of morbidity; ICD-10 NA, the Application to Neurology, is a detailed classification of diseases of neurological origin or expression, based on the original ICD-10. Thus it also contains a table of inclusion and exclusion terms and a detailed alphabetical index. The inclusion terms are names of diseases, affections or syndromes associated with each ICD code. The exclusion terms are listed so that the disease in question is classified elsewhere. Thus it is easy to find the correct category even for similar or related diseases which may be classified in different chapters of the ICD-10 NA. The coding system of the ICD-10 NA is exactly the same as for the ICD-10 since cross-referencing requires that specialization classifications such as the ICD-10 NA derived from the ICD must have the same 3 and 4 digit codes as those in the original ICD-10. Within these limits, the 5th, 6th and 7th digits of the subdivision codes, and the generalization of multiple coding used to describe both aetiologies and manifestations, have greatly increased the discriminating power and precision of neurological diseases classification based on the parent ICD.

Twin pregnancies in the second trimester in women in an alpha-fetoprotein screening program: sonographic evaluation and outcome.

OBJECTIVE: We correlated sonographic findings with fetal outcomes in women with unsuspected twin pregnancies who had sonography in the second trimester as part of a screening program for maternal serum alpha-fetoprotein (MSAFP) level and history of neural tube defect. MATERIALS AND METHODS: The study group consisted of 97 women with twin pregnancies who participated in a screening program for MSAFP level and history of neural tube defect. Seventy-three had normal MSAFP levels, 21 had elevated MSAFP levels, and two had low MSAFP levels. One patient had a family history of anencephaly. All 97 patients had sonography during their second trimester of pregnancy. Sonographic findings were reviewed retrospectively for information on gestational age, fetal anomalies, sex of the fetus, location of the placenta, presence and thickness of a dividing membrane, and interpretation of amnionicity and chorionicity. Information on fetal outcome included gestational age at delivery, survival, birth weight, sex, congenital anomalies, obstetric complications, amnionicity, chorionicity, and placental abnormalities. RESULTS: Amnionicity and chorionicity were correctly detected on sonograms in 44 (90%) of 49 diamniotic-dichorionic gestations, 23 (72%) of 32 diamniotic-monochorionic gestations, and two (50%) of four monoamniotic-monochorionic gestations. Fetal anomalies were present at delivery in five neonates and had been correctly detected at sonography in one (hemivertebra); one fetus with duodenal atresia had abnormal sonographic findings in the third trimester. Missed anomalies included absent forearm, cleft lip and palate, and imperforate anus. Sex of the fetuses was correctly predicted on the basis of sonographic findings in 40 of 43 pairs. Nine twin pairs had possible twin-twin transfusion syndrome suspected sonographically on the basis of abnormal fluid volumes, discrepant growth measurements, and abnormal findings on Doppler studies. Outcomes included two confirmed cases of the syndrome (two survivors, two deaths) and three probable cases (six deaths); four pregnancies resulted in eight survivors who were delivered after 34.4 weeks' gestation and had birth weights in the 25th percentile or higher. Survival rates for diamniotic-dichorionic, diamniotic-monochorionic, and monoamniotic-monochorionic gestations were 90%, 91%, and 50%, respectively. Fetuses in women with MSAFP levels greater than 4.5 multiples of the median and with monochorionic placentation had lower survival rates than fetuses in women with normal MSAFP levels and monochorionic placentation (67% vs 96%). Half the fetuses delivered after 20 weeks' gestation had birth-weight discordance of less than 10%. Premature deliveries occurred in 56% of pregnancies. CONCLUSION: The results suggest that (1) sonography is useful in predicting placentation, (2) placentation may be helpful in predicting fetal outcome, (3) increased MSAFP levels correlate with increased perinatal mortality in diamniotic-monochorionic pregnancies, and (4) caution should be taken in diagnosing and determining prognosis for suspected twin-twin transfusion syndrome in the second trimester.

Occluding staghorn calcification of the aortic arch and left subclavian artery: case report.

A calcified thrombus resembling a staghorn was found in the aortic arch, cast into this shape by the aorta and its branching vessel, the left subclavian, into which it projected for a short distance. Unique, in this case, is the extraordinarily large size of the calcification which was mostly free of the vessel wall, its radiological image, and its location. Digital subtraction angiography led to the diagnosis and was confirmed on computed tomography.

Short-term effect on growth of two doses of GRF 1-44 in children with growth hormone deficiency: comparison with growth induced by methionyl-GH administration.

In a double-blind study 12 prepubertal children with idiopathic growth hormone (GH) deficiency were treated with growth hormone releasing factor (GRF) 1-44 in a dosage of 7.5 or 15 micrograms/kg body weight, administered once a day subcutaneously. With 7.5 micrograms/kg the average growth velocity increased from 2.5 to 4.6 cm/year, an insufficient response. With the higher dosage the average growth velocity increased from 2.7 to 7.0 cm/year, a similar increase as observed with GH therapy in subsequent periods. In 3 of the 6 children treated with the higher dose appropriate catch-up growth was observed. The growth response of the lower leg length was not always consistent with the statural growth response.