大鼠體內L-3-羥基丁酸之分析及其在心臟之作用
Determination of L-3-Hydroxybutyrate in rats and its effects on rat heart
中文摘要
D-3-Hydroxybutyrate（D-3HB）為體內之 ketone bodies 中含量最高者，且被作為 研究ketone bodies 之作用的主要目標。相反地，一般認為 L-3-
hydroxybutyrate（L-3HB）並非生物體內源生之 ketone body，或許是基於其尚有 爭議的代謝途徑；以及目前不瞭解其生成來源。在本論文中以螢光衍生化法搭配 High-Performance Liquid Chromatography（HPLC）所開發之分析方法已能證實 rat serum 中的確有 L-3HB 的存在。Serum 中之 total 3HB 經 NBD-PZ 衍生化後先 經由一ODS column 將之分離，隨後以兩支 CHIRALCEL OD-RH 串聯之 chiral columns 進行 chiral separation。Rat serum 並以 D-3-hydroxybutyryl dehydrogenase
處理作為對照組，以驗證所分離之3HB 的真實性。實驗結果顯示 serum 中含有
L-3HB，其與 D-3HB 的濃度分別為 3.98（3.61%）與 106.20 M（96.39%）。在
以此分析方法成功地證實L-3HB 存在後，我們再進一步應用於 rat 體內各組織
中D-與 L-3HB 的分佈情形。
分取rat 之腦、肝、心、及腎的研磨後均質液，經分析後發現 heart 中含有特殊高量
的L-3HB；為所有檢測的組織中最特別者。D/L-3HB 之比例在正常與 diabetes mellitus（DM）時有所不同，其比例（D/L）分別為 66/37 與 87/13。此變化可能 是造成glucose 代謝能力受影響的原因之一。當投予 5 mM 之 D-3HB 於 medium 中，cardiomyocytes 的 glucose 代謝會降低至控制組的 61%，但給予同等劑量之 L-3HB 並未對細胞之 glucose 代謝造成任何影響，此結果反映著 L-3HB 與其他 ketone bodies 不同；並非作為提供能量的物質。此外，D-3HB 抑制 glucose 代謝 的作用可被另行添加之L-3HB 阻斷；且 L-3HB 回復 glucose 代謝的作用會隨著 其濃度增加而升高。藉由測量cardiomyocytes 代謝 D-、L-、與(D+L)-3HB 能力的 結果發現，D-與 L-3HB 同時存在的情況下反而會加速 D-3HB 的代謝；且可發 現L-3HB 的生成。由此推測 L-3HB 會刺激 D-3HB 進行 interconversion 生成 L- 3HB；使 D-3HB 的代謝不再回復產生 acetyl CoA。實驗結果顯示 L-3HB 有別於 D-3HB，為維持正常 glucose utilization 的重要物質，其可能在 ketone bodies 與 glucose 的代謝之間扮演著調節的角色。
英文摘要
While D-3-Hydroxybutyrate (D-3HB) is usually the major ketone body which was under intensive investigation, little attention had been paid to L-3-hydroxybutyrate (L-3HB). It had been considered nonexistent physiologically, perhaps due to its dubious metabolic route and lack of knowledge about its origin. In the present study, we proved that L-3HB is an original substance in rat serum by applying fluorescence
derivatization and a column-switching high-performance liquid chromatography (HPLC) system as the analysis technique. Total 3HB in rat serum derivatized by 4- nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was separated by an ODS
column, and was confirmed by verifying the disappearance of the total 3HB peak after pretreating rat serum with D-3-hydroxybutyryl dehydrogenase (D-3HB
dehydrogenase). A switching valve was used to simultaneously introduce isolated (D+L)-3HB to the enantiomeric separation by two CHIRALCEL OD-RH columns connected in tandem. An L-isomer was found to accompany the D-isomer, which were quantified to be 3.98 (3.61%) and 106.20 M (96.39%), respectively. Using the present analytical method, the dubious interpretation of the existence of L-3HB was clarified.
Subsequently, distribution of D- and L-3HB in rat brain, liver, heart, and kidney homogenates were measured. The results showed that an enriched amount of L-3HB is present in rat hearts. The ratio would be changed from 66/34 to 87/13 (D/L) in normal and diabetic states, respectively. The altered D/L ratio may contribute to the reduction in glucose utilization by cardiomyocytes. Glucose utilization of
cardiomyocytes with 5 mM of D-3HB was decreased to 61% of the control, but no interfering was observed when D-3HB was replaced with L-3HB, suggesting L-3HB is not utilized as the energy fuel as other ketone bodies are. In addition, the reduced glucose utilization caused by D-3HB could gradually recover in a dose-dependent manner with administration of additional L-3HB. Determination on metabolism of D-, L-, and (D+L)-3HB by cardiomyocytes showed cells had increased D-3HB
metabolism when (D+L)-3HB was administered, and re-generation of L-3HB was found under the circumstance. It was speculated that in the presence of L-3HB, D- 3HB might go through interconversion to generate L-3HB rather than being oxidized to acetyl CoA. The results suggest that it is a necessity of taking L-3HB together with D-3HB when it comes to glucose utilization. A physiological role is proposed for L- 3HB as an important substrate which regulates the metabolism between glucose and ketone bodies.
