Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

Upon absorption in the intestine of the host animal, the main function of short-chain fatty acids (SCFAs), mainly acetate, propionate and n-butyrate, is as metabolic energy. SCFAs, n-butyrate in particular, can also be found in the mouth. An excess of oral SCFAs may cause not only periodontal diseases but also systemic abnormalities in humans. Previously, we reported a method for simultaneous detection by gas chromatography-mass spectrometry (GC-MS) of acetate, propionate and n-butyrate in serum, urine and saliva. In the present study we used a modified version of this method to detect not only acetate, propionate and n-butyrate, but also iso-butyrate, n-valerate, iso-valerate and caproate, because the latter are suggested to be associated with periodontal diseases. Detection ranges of SCFAs were as follows; 6.25-400 µmol/L (acetate), 0.781-100 µmol/L (propionate and n-butyrate), 0.391-50 µmol/L (iso-butyrate), 0.781-50 µmol/L (n-valerate and iso-valerate) and 1.56-50 µmol/L (caproate). Furthermore, we validated the modified detection method with triple freeze-thaw-cycle recovery tests and intra-and inter-day repeatability. Freezing and thawing did not influence the concentrations of SCFAs in saliva. Upon analysis of five clinical saliva samples, it was observed that, except for n-valerate, which was detected only in two samples, all SCFAs were detected in saliva samples. To conclude, we were able to use a modified method to analyze successfully by GC-MS the salivary concentrations of SCFAs. In addition, we simultaneously detected the salivary concentrations of iso-butyrate, iso-valerate, nvalerate and caproate. This improved method was proved to be reliable to measure the concentrations of SCFAs in saliva.

Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at Ileu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run < 4.45%) and a coefficient of correlation of r = 0.985 (N = 125).

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