大鼠體內 L-3- 羥基丁酸之分析及其在心臟之作用

1. 大鼠體內L-3-羥基丁酸之分析及其在心臟之作用大鼠體內L-3-羥基丁酸之分析及其在心臟之作用 • 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。

2. Determination of L-3-Hydroxybutyrate in rats and its effects on rat heart • 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.