Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by (1)H NMR spectroscopy and PLS regression.

Danaparoid sodium (the active pharmaceutical ingredient in Orgaran; Merck Sharp and Dohme) is a biopolymeric non-heparin drug used as anticoagulant and antithrombotic agent approved for the prophylaxis of post-operative deep-vein thrombosis, which may lead to pulmonary embolism in patients undergoing, e.g., elective hip replacement surgery. It consists of a mixture of three glycosaminoglycans (GAGs): heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Currently, the CS and DS content are quantified by means of a time-consuming enzymatic method. In this paper the use of (1)H NMR in combination with multivariate regression (partial least-squares, PLS) is proposed as a new method. In order to evaluate the proposed method, a series of danaparoid sodium samples were analyzed and the results were compared with those obtained by the enzymatic method (reference method). The results showed that the proposed (1)H NMR method is a good alternative for analysis of CS and DS in danaparoid sodium. Accuracy of ±0.7% (w/w) and ±1.1% (w/w) for CS and DS was obtained by the (1)H NMR method and accuracy of ±1.0% (w/w) and ±1.3% (w/w) by the enzymatic method. Furthermore, the use of (1)H NMR in combination with PLS results in a fast quantification. The analysis time is reduced to 35 min per sample instead of 60 h for a maximum of 16 samples.

Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.

Sodium influxes in renal epithelial LLC-PK1/Cl4 cells monitored by 23Na NMR.

23Na NMR experiments with a suspension of the renal epithelial cell line, LLC-PK1/Cl4, in the presence of the shift reagent dysprosium polyphosphate showed that the intracellular sodium was only NMR visible for 64 +/- 4%. Intracellular sodium content was found to be 30.6 +/- 1.2 mM (25 degrees C). Examination of the sodium influx during recovery from intracellular acidification showed that sodium is transported not only by Na+/H+ exchange but also by sodium-D-glucose cotransport with a stoichiometry of 1:1.

Dose-response relationships of the tumorigenicity of cyclopenta[cd]pyrene, benzo[a]pyrene and 6-nitrochrysene in a newborn mouse lung adenoma bioassay.

Cyclopenta[cd]pyrene (CPP) was a potent tumorigen when tested over a 5-fold dose range in the newborn mouse assay. A 20-fold increase in lung tumor multiplicity and a nearly 8-fold increase in tumor incidence was observed at the lowest total dose tested (1.55 mumol) with the dose-response relationship indicating a saturation of tumor multiplicity at approximately 7 tumors/animal. No liver nodules or lymphatic system tumors were noted. Analysis of dose-response data for benzo[a]-pyrene (BaP) and 6-nitrochrysene (6-NC) showed that tumor multiplicity for BaP also saturated at approximately 7 tumors/animal, whereas no similar saturation was found for 6-NC at up to 40 tumors/animal. Progression of lung adenomas to adenocarcinomas, as measured by the incidence of mice bearing malignant tumors, was essentially a linear function of dose. To facilitate comparison and maximize quantitative data obtainable from the newborn mouse assay-parameters were defined for tumor incidence (ED50), tumor multiplicity (TM1.0) and tumor malignancy (malignancy index). Values for the ED50 and TM1.0 were similar for the same compound and a tumorigenic potency series of 6-NC greater than BaP greater than CPP was obtained corresponding to a ratio of approximately 1:10-25:76.5-135, respectively. The malignancy index, however, indicated increased adenocarcinoma induction in the order CPP greater than 6-NC greater than BaP as expressed by the ratio 1:1.4:8.3, respectively.