Raymond A. Smith

Effect of sodium saccharin and calcium saccharin on urinary parameters in rats fed Prolab 3200 or AIN-76 diet

The effects of the salt form of saccharin and of diet on urinary ion levels have been studied in rats. Sodium saccharin (NaS) or calcium saccharin (CaS) was fed at a level of 5% in either Agway Prolab 3200 diet or AIN-76 diet to male, 5-wk-old F344 rats for 10 wk. The AIN-76 diet contained considerably less calcium, sodium and potassium than the Prolab 3200 diet, and smaller amounts of these ions were eliminated over 24 hr in the urine of rats fed the AIN-76 diet. Although food consumption was less in the groups fed AIN-76, total urinary saccharinate ion excretion with either saccharin salt was comparable with, or even higher than, that excreted by rats fed either salt in the Prolab 3200 diet. Rats fed Prolab 3200 eliminated approximately equal amounts of saccharinate ion in the faeces and urine. Rats fed AIN-76 eliminated about 10-20 times as much saccharin in the urine as in the faeces. Total saccharin excretion (faecal and urinary) was not influenced by the salt form. Water intake and urine volume were lower in rats fed control AIN-76 diet in comparison with those fed Prolab 3200, and were increased above the control level in groups fed saccharin in the AIN-76 diet. Urine electrolyte levels and osmolality were lower in the groups fed AIN-76. In general, NaS administration in either diet resulted in increased urinary sodium compared with controls, and the pH was at, or above, the level of control rats. CaS resulted in increased urinary calcium and decreased pH. There were marked diurnal variations in the urinary excretion of the various electrolytes, pH, and urine volume over a 24-hr period in all rats. This diurnal variation was more pronounced in the rats fed the Prolab 3200 diet. These results indicate that NaS and CaS have marked effects on the excretion of urinary electrolytes, and that these effects are influenced by diet.

Phase-sensitive heteronuclear multiple-bond correlation in the presence of modest homonuclear coupling. Application to distamycin A

Long-range correlation of proton and heteronuclear chemical shifts, in particular carbon, has proven invaluable in the investigation of mo lecular structure ( I7). The advent of indirect detection of heteronuclei via protons has made long-range correlation routine even on low concentration samples. The most useful indirect technique for long-range correlation proposed thus far is the heteronuclear mu ltiple-bond correlation (HMBC) experiment ( 7). We have used this technique with excellent results despite the fact that we use the proton-decoupl ing coil of our standard Varian 5 m m broadband switchable probe for observation. Typically, we achieve a nominal fivefold increase in signal-to-noise ratio (S/N) per unit measur ing time compared to the 13C detected experiment. We have recently encountered difficult assignment problems via work with analogs of the DNA-binding drug distamycin A (8-l 1) . These problems are due to low sensitivity and poor proton resolution. Distamycin A, for instance, shows a significant degree of aromatic proton overlap in its ‘H spectrum (Table 1) . Also, complete assignment of the 13C and ‘H spectra requires correlation of carbonyl resonances with aromatic ring protons via 4 JCH which is quite small, requiring the highest sensitivity possible. The sensitivity of HMBC has been addressed by Bax and Marion ( 12). They proposed a m ixed mode data-processing procedure for the HMBC experiment, which utilizes a hypercomplex Fourier transform (13) of the data, resulting in absorptive mode in F, followed by a magn itude calculation in F2. This technique results in a \Iz increase in S/N. The reason for applying a F2 magn itude calculation to the HMBC data in both the conventional method and Bax and Marion’s experiment is the presence of phase modu lation of the F2 data by proton chemical shift and homonuclear coupling during the delay, 7, in F ig. la. Since distamycin A and its congeners show only modest (e.g., 2 Hz) scalar coupling in the over lapped aromatic ‘H region, it seemed that the m inimal amount of phase modu lation due to these couplings m ight

Quantitation of uracil in rodent diet

Feeding of high levels of uracil to laboratory rodents results in the formation of calculi in the lumen of the urinary bladder. This urolithiasis stimulates cellular proliferation in the bladder and has been used in studies of two-stage carcinogenesis. Quantitation of uracil in rodent diet was achieved by extraction from the diet with ammonium hydroxide. The extract was applied to a strong anion-exchange solid-phase extraction column. Uracil is not retained on this matrix which adsorbs the majority of contaminants in the extract. The uracil was quantitated by HPLC on an ODS microbore column (100 x 2 mm internal diameter) eluted at 0.5 ml/min with 200 mM KH2PO4, pH 3.5, at 30 degrees C. Three structurally related pyrimidine bases, cytosine, uracil, and thymine, showed increasing retention on this column/solvent combination, thereby demonstrating selectivity of the analysis. Recovery of uracil was 76-90% with lower values observed when dietary levels of uracil were in excess of 4.5%.