Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.     

The first metal complexes bearing ligated 3-iminoisoindolin-1-ones

The reaction between Zn(OAc)₂ · 2H₂O (1) and the 3-iminoisoindolin-1-ones H₂NCNC(O)C₆R¹R²R³R⁴ (R¹-R⁴ = H 2; R¹, R⁴ = H, R², R³ = Cl 3; R¹, R³, R⁴ = H, R² = Me 4) in EtCN at 70 °C for ca. 12 h affords the novel family of complexes [Zn{H₂NCNC(O)C₆R¹R²R³R⁴}₂(OAc)₂] (R¹-R⁴ = H 5; R¹, R⁴ = H, R², R³ = Cl 6; R¹, R³, R⁴ = H, R² = Me 7) in excellent (90% and 93% for 5 and 6, correspondingly) to good (64% for 7) yields. The isolated compounds were characterized by elemental analyses (C, H, N), IR, NMR and ESI⁺-MS. X-ray diffraction data for 2 and 5 indicate that both free (2) and ligated (5) 3-iminoisoindolin-1-ones exist in the zwitterionic form.     

Modifications of drug metabolism by disulfiram and diethyldithiocarbamate. II. D-Glucuronic acid pathway.

Hepatic enzymes connected with the formation and metabolism of free D-glucuronic acid were affected in rats after treatment with disulfiram or diethyldithiocarbamate (300 mg/kg, intragastrically, per day, 4 X). The activities of UDPglucose dehydrogenase, UDPglucuronic acid pyrophosphatase, UDPglucuronosyltransferase and L-gulonate dehydrogenase were enhanced, while those of glucose-6-phosphate dehydrogenase, beta-glucuronidase and D-glucuronolactone dehydrogenase were inhibited. These changes were more pronounced with disulfiram than diethyldithiocarbamate. Treatment with phenobarbital (80 mg/kg, i.p., per day, 4 X) enhanced UDP glucuronosyl-transferase, but brought about different effects on the other enzymes. Concurrent administration of phenobarbital with disulfiram or diethyldithiocarbamate led to potentiation or antagonism of the primary effects of each compound when given alone. The results suggest that activation of the D-glucuronic acid pathway may proceed in various ways, and that it is not necessarily followed by a simultaneous induction of the microsomal mixed-function oxygenase activity.     

Quantitative determination of dehydroepiandrosterone fatty acyl esters in human female adipose tissue and serum using mass spectrometric methods

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) are naturally occurring water-insoluble metabolites of DHEA, which are transported in plasma exclusively by lipoproteins. To find out whether DHEA, like estradiol, might be stored in adipose tissue in FAE form, we set up a mass spectrometric method to quantify DHEA-FAE and free DHEA in human adipose tissue and serum. The method consists of chromatographic purification steps and final determination of hydrolyzed DHEA-FAE and free DHEA, which was carried out by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that no detectable amounts of DHEA-FAE could be found in adipose tissue although 32-178 pmol/g of free DHEA were determined by GC-MS and LC-MS/MS. The DHEA-FAE concentrations in serum quantified by GC-MS were 1.4±0.7 pmol/ml in premenopausal women (n=7), and 0.9±0.4 pmol/ml in postmenopausal women (n=5). Correspondingly, the free DHEA concentrations were 15.2±6.3 pmol/ml and 6.8±3.0 pmol/ml. In addition, the mean proportions of DHEA-FAE of total DHEA (DHEA-FAE+free DHEA) in serum were 8.6% and 11.2% in pre- and postmenopausal women, respectively. Serum DHEA-FAE concentration was below quantification limit for LC-MS/MS (signal-to-noise ratio, S/N=10), while free DHEA concentrations varied between 5.8 and 23.2 pmol/ml. In conclusion, the proportion of DHEA-FAE of total DHEA in serum was approximately 9%. However, in contrast to our previous findings for estradiol fatty acid esters in adipose tissue which constituted about 80% of total estradiol (esterified+free), the proportion of DHEA-FAE of total DHEA was below 5%. Four to ten times higher concentrations of free DHEA were quantified in adipose tissue compared to those in serum.     

Prosthetic crowns and other clinical risk indicators of caries among old-old Swedish adults: findings from the KEOHS Project. Kungsholmen Elders Oral Health Study

Objectives: The Kungsholmen Elders Oral Health Study (KEOHS) evaluated the oral health status of generally healthy, community‐dwelling persons over the age of 80 living in Kungsholmen, Sweden. This paper explored possible clinical risk indicators of coronal and root caries among the KEOHS subjects. Design: In this cross‐sectional study, dentate KEOHS subjects received a caries assessment using defined visual, tactile criteria. Setting: Examinations were carried out in two local clinics by standardized examiners. Subjects: One hundred twenty‐nine dentate persons were examined. Main Outcome Measures: The examination identified decayed and filled surfaces, prosthetic crowns, and missing teeth. Results: More root than coronal surfaces had untreated decay, and secondary root caries contributed the greatest number of decayed surfaces. Ninety percent of the examined dentate subjects had at least one prosthetic crown. Root surfaces exposed to crown margins were more likely to have caries than root surfaces not so exposed, particularly among women. The presence of untreated coronal caries (yes/no) was positively associated with having untreated root caries and an intermediate number (14–20) of teeth, but inversely associated with having 4+ prosthetic crowns. Active root caries (yes/no) was positively associated with having untreated coronal caries, 14–20 teeth, and 4+ prosthetic crowns. Nearly 20% of identified root lesions were present at or below the gingival margin, and most (88%) were secondary caries associated with crown margins (65%) or other restorations (23%). Conclusions: Our findings suggest that some dental characteristics, including the presence of prosthetic crowns, are risk indicators for the presence of untreated coronal and root caries.     

Comparison of gas chromatography-mass spectrometry and high-performance liquid chromatography with coulometric electrode array detection for determination of alkylresorcinol metabolites in human urine

Alkylresorcinols (AR) are amphiphilic compounds present at high concentrations in the outer parts of wheat and rye kernels. Due to their specificity to whole grain and bran products of these cereals, AR and their metabolites have been proposed as biomarkers for intake of such foods. Two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), have previously been quantified in human urine using two different methodologies: high-performance liquid chromatography coupled to a coulometric electrode array detector (HPLC-CEAD) and gas chromatography in combination with mass spectrometry (GC-MS). In this study, these two methodologies were compared by analysing 114 urine samples from free-living Swedish subjects consuming their habitual diet. Data were evaluated by graphical investigation of difference-plots and statistical inference of agreement was assessed by weighted Deming regression analysis. The median DHBA concentrations were 11 μM (GC-MS) and 13 μM (HPLC-CEAD), respectively. Both difference-plot and regression analysis showed a small but statistically significant additive bias, with HPLC-CEAD resulting in a slightly higher DHBA concentration than GC-MS. The median concentration of DHPPA was 18 μM for both methods. Examination of the difference-plot of DHPPA did not indicate any systematic difference between the methods, but regression analysis showed small but statistically significant constant and proportional biases. The conclusion was that the two methodologies are equally suitable for analysis of alkylresorcinol metabolites in human urine and that any small systematic differences observed are most likely of limited practical importance.     

Leukemia Prediction Using Sparse Logistic Regression

We describe a supervised prediction method for diagnosis of acute myeloid leukemia (AML) from patient samples based on flow cytometry measurements. We use a data driven approach with machine learning methods to train a computational model that takes in flow cytometry measurements from a single patient and gives a confidence score of the patient being AML-positive. Our solution is based on an regularized logistic regression model that aggregates AML test statistics calculated from individual test tubes with different cell populations and fluorescent markers. The model construction is entirely data driven and no prior biological knowledge is used. The described solution scored a 100% classification accuracy in the DREAM6/FlowCAP2 Molecular Classification of Acute Myeloid Leukaemia Challenge against a golden standard consisting of 20 AML-positive and 160 healthy patients. Here we perform a more extensive validation of the prediction model performance and further improve and simplify our original method showing that statistically equal results can be obtained by using simple average marker intensities as features in the logistic regression model. In addition to the logistic regression based model, we also present other classification models and compare their performance quantitatively. The key benefit in our prediction method compared to other solutions with similar performance is that our model only uses a small fraction of the flow cytometry measurements making our solution highly economical.