Kenji Shimada

Characterization of rat myocardial sarcolemma

Sarcolemma (SL) was isolated from the hearts of rats by the procedure of Philipson et al. and was found to be of high purity by the use of electron microscopy and marker enzyme analysis. In the SL fraction the sarcolemmal marker enzyme Na+, K+-ATPase was enriched about 33-fold (without SDS stimulation) compared to the crude homogenate. The SL phospholipid (PL) specific content was 0.76 μmol mg−1 SL protein and although this is about three-fold higher than that previously seen in the rat, it is substantially lower than the [PL] in SL reported by others isolated from other mammals. The SL cholesterol was almost entirely non-esterified and the cholesterol: PL molar ratio was 0.547. The individual PL were separated by thin-layer chromatography (TLC). Sarcolemmal PL from the rat have a distribution that is distinct from that observed by others in both rabbit and dog hearts. Although phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) together accounted for slightly more than 70% of the total PL as in other mammals, the PCPE ratio in the rat was 0.94 and is substantially lower than the 1.21 to 1.40 range observed by others in rabbit and dog hearts. The anionic PL phosphatidyl serine (PS), known to be an important component of SL Ca2+ binding, had a concentration in the rat of about 40% of the [PS] of other mammalian SL. Conversely, sphingomyelin was found in significantly higher concentrations in the rat. The SL fatty acid composition was also determined and was significantly more saturated than the SL from rabbit heart. The significance of the differences in rat SL composition compared to other mammals is not known but may be related to its profoundly different E-C coupling characteristics.

Fluorometric high-performance liquid chromatography of 9-aminophenanthrene-derivatized free fatty acids

The application of 9-aminophenanthrene (9-AP), a fluorescence-labeling reagent for free fatty acids (FFA), was examined. 9-AP dissolved in benzene was added to a benzene solution of FFA chlorides derived from FFA and oxalyl chloride. The mixture was allowed to react for 45 min at 70 degrees C. By the method, 9-AP-tagged FFA with a strong fluorescence was formed. The materials thus obtained have a lambda max at around 303 nm for excitation and 376 nm for emission. By using this derivatization method, recoveries were measured for seven kinds of FFA added to 0.5 ml of healthy human serum. Significant recoveries ranging from 96 to 107% (coefficient of variation 1.4-5.0%) were obtained for each FFA. The proposed method was clinically applied to the determination of FFA in 0.5 ml of healthy human serum, and almost satisfactory results were obtained. Detection limits of FFA by this derivatization method were 10 pmol for C14:0, C16:0, C16:1, C18:1 and C18:2, and 15 pmol for C18:0 and C20:4. As a quantitative measurement of FFA, gas chromatography and high-performance liquid chromatography with fluorescence detection, which have been routinely used, were chosen for comparison with the present method.

High-performance liquid chromatographic determination of free fatty acids with 1-naphthylamine

A 1% benzene solution of oxalyl chloride was added to saturated and unsaturated fatty acids and the mixture was allowed to react at 70 degrees C for 30 min; by this procedure, each fatty acid was converted into its acid chloride in a considerably quantitative manner. By reacting this acid chloride with 1-naphthylamine at 30 degrees C for 15 min, naphtylamine derivatives were produced, which showed strong ultraviolet absorption around 280-290 nm. Experiments were made on the recovery of the fatty acids added to 0.5 ml of human serum, and the recovery was found to fall in the range of 94-106% (coefficient of variation = 0.5-4.1%) when the following amounts of six fatty acids were added: C14:0, 2 micrograms; C16:0, 20 micrograms; C16:1, 5 micrograms; C18:0, 4 micrograms; C18:1, 20 micrograms; C18:2, 10 micrograms.