| Summary: | 博士 === 中山醫學大學 === 醫學研究所 === 97 === Backgrounds
Little is known about the association between human haptoglobin phenotypes and diseases. Also, not much is known about the association between blood chitinolytic enzyme levels and human diseases. Recent studies have suggested a link between haptoglobin and the molecules associated with inflammation. Subsequent discoveries on its roles in T-cell development and the governing signaling pathways have further enlightened both basic and epidermiological studies. The understanding of human chitinases, on the other hand, is relatively limited. To our knowledge, lacking of rapid and convenient analytical methods has limited the progress on studying both molecules in human.
Our study aimed to develop rapid and accurate biochemical methods in analyzing (1) the haptoglobin phenotypes in human plasma, and (2) the specific activity of chitinolytic enzymes. These analytical methods were then applied to plasma samples from clinical and general populations in investigating the possible associations between human diseases and these traits.
Methods
We have developed a new method, termed mCBB-R250 staining, for rapid and sensitive typing of haptoglobin phenotypes from human plasma. The percentage of frequencies and the proportion of the three haptoglobin phenotypes, Hp 1-1, Hp 2-1, and Hp 2-2, in our collection of human plasma samples were analyzed using descriptive statistic, one sample t test and Chi-square tests.
Using glycol chitosan as substrate, an analysis method using (K3Fe(CN)6) as indicator were developed to determine the chitioson-degrading enzymes in human plasma. An in-gel activity staining method was also developed for the detection and separation of chitinolytic enzymes. The specific activities of chitinolytic enzymes were analyzed using the normality test, independent-sample parametrical test, or non-parametrical test.
Results
We have successfully applied the mCBB-R250 method to determine the Hp phenotypes of 1148 samples. Among these, 11.67% are Hp 1-1, 42.60% are Hp 2-1, and 45.73% are Hp 2-2 type. As a comparison, the distribution of the three Hp phenotypes, as determined for 151 plasma samples from patients with cardiovascular diseases, are 11.26%, 41.06%, and 47.68% respectively for Hp 1-1, 2-1, and 2-2 type. These percentages are similar to that of the general population. Among the 151 samples from cardiovascular disease patients, 101 are stable angina (CAD), and the rest 50 are acute coronary syndrome (ACS) patients. While the percentages of Hp 2-2 type are statistically indifferent between these groups, the percentages of the Hp 1-1 and Hp 2-1 types are significantly different among CAD and ACS groups, in which we found that Hp 2-1 type is higher in CAD, but Hp 1-1 is higher in ACS.
Using glycol chitosan and (K3Fe(CN)6) solution, the specific activities of chitinoltic enzymes, after analyzing 899 human plasma samples, was determined to be a normal distribution, with an average specific activity of 92.00 U/mg (95% CI 90.584，93.416). We have found that, among 151 patients of cardiovascular diseases, the specific chitinolytic enzyme activities in CAD group (102.724 U/mg) are higher than those in the ACS group (90.513 U/mg). We have also developed an in-gel detection method for rapid separation and detection of chitinolytic enzymes. With this new method, we have identified a new chitinolytic enzyme with a molecular weight of about 30 kDa.
Conclusion
We have developed rapid, accurate, and inexpensive methods for haptoglobin phenotyping and chitinolytic enzyme activity staining. The methods were applied to the Hp phenotyping and chitinolytic enzyme activity detection of 1178 plasma samples. With their application in clinical studies, these methods may grant us further understandings on the possible association with human diseases.
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