Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio-mass spectrometric analysis.

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.

Separation of polyunsaturated fatty acids by selective inclusion in guanidinium-1,5-naphthalenedisulfonate-2-methoxyethanol host networks.

Hydrogen-bonded networks composed of guanidinium (G) and 1,5-naphthalenedisulfonate (NDS) have been developed for the selective inclusion and separation of fatty acid esters based on their degree of unsaturation. Porous crystalline networks have been synthesized and include fatty acid esters during crystallization from both methanol and 2-methoxyethanol. Crystalline networks formed in methanol are selective for the inclusion of saturates in preference to polyunsaturated fatty acid methyl esters, but their applicability is limited by the competing inclusion of the solvent methanol. In 2-methoxyethanol, a three-component host structure is formed that provides 4.1 x 4.7 A2 pores which are also selective for saturated fatty acid ester inclusion. These networks do not suffer from the competing inclusion of the solvent 2-methoxyethanol as is the case with methanol, and thus complete removal of initial saturated fatty acid 2-methoxyethyl esters is possible. Binary selectivity experiments on mixtures of the 2-methoxyethyl esters of saturated stearic acid and the omega-3 fatty acid alpha-linolenic acid reveal that this three-component network structure provides nearly complete resolution of these two guests in the crystal and filtrate fractions. Separation of the 2-methoxyethyl esters of alpha-linolenic acid from the monounsaturated oleic acid is also possible, although with decreased efficiency in comparison to removal of the saturated fatty acid ester.