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성장기 흰쥐의 골조직 Collagen 생성속도 측정

Measuring in vivo Rate of Bone Collagen Synthesis in Growing Rats

  • 발행 : 2003.12.01

초록

동위 원소를 이용하여 골조직 collagen과 같이 대사속도가 느린 단백질의 합성 속도를 측정 할 때 가장 중요한 점은 전구체 공급원의 비균일성 및 복잡한 대사경로로 인한 해석상의 제한점을 극복하기 위하여 동위원소가 함유된 전구체를 지속적으로 투여하여 동위원소공급원의 항상성을 유지하는 것이다. 이 경우 precursor-product kinetics를 적용하여 $k_{s}$값을 정확하게 측정할 수 있다. $^2$$H_2O$는 저렴한 안정동위 원소 추적자로서 독성이나 부작용 없이 장기간 공급 가능하고 공급방법 또한 간단하다. 본 연구에서는 골조직 collagen 합성속도를 아미노산에 $^2$$H_2O$를 치환하는 precursor-product 방법으로 측정하였으며, 골조직 collagen으로부터 분리한 아미노산의 동위 원소함량을 GC/MS로 분석하였다. 실험 기간동안 체액의 $^2$$H_2O$는 2.7 ∼ 3.0% 수준으로 일정하게 유지되었고 성장기 흰쥐 골조직의 collagen 합성속도는 $k_{s}$(OH-pro) = 0.066$\pm$0.049 w $k^{-1}$였다. 동위원소를 연속적으로 공급해야하는 precursor product방법은 교체율이 느린 단백질에 적용하는데 기술적인 어려움이 있었지만, 안정하고 저렴한 $^2$$H_2O$ 특성으로 인하여 precursor-product방법을 교체율이 느린 단백질에 적용하는 것이 가능하게 되었다..

Measuring in vivo rate of bone collagen synthesis has so far been technically difficult and often subject to quite large errors. In the present study, bone collagen synthesis rate was measured using a precursor-product method, based on the exchange of $^2$$H_2O$ into amino acids. Mass isotopomer abundance in hydroxyproline from bone collagen was analyzed by gas chromatography/mass spectrometry. The $^2$$H_2O$ labeling protocol consisted of an initial intraperitoneal injection of 99.9% $^2$$H_2O$, to achieve approximately 2.5% body water enrichment followed by administration of 4% $^2$$H_2O$ in drinking water for 9 weeks. Body $^2$$H_2O$ enrichments were stable at 2.7 ∼ 3.0% over labeling Period. In growing rats, the fractional synthesis rate ( $k_{s}$) of bone collagen was 0.066 $\pm$ 0.049 w $k^{-1}$ . The unique features of stable $^2$$H_2O$ pools and label incorporation allowed the precursor-product approach to be used for measuring bone collagen synthesis rate..

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참고문헌

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