■血管紧张素-(1-12),以自我开发的方式衡量,基于多克隆抗体的放射免疫分析,已被建议作为血管紧张素II的替代前体。更可靠的检测方法是液相色谱-串联质谱法。
■我们通过液相色谱-串联质谱法建立了人和鼠血管紧张素-(1-12)的定量,然后使用该方法测量了人和小鼠血液样品中的血管紧张素-(1-12),以及老鼠的大脑和肾脏。我们还验证了在37°C孵育的人血液样品中的离体血管紧张素-(1-12)生成和代谢。
■选择血液在盐酸胍中的稳定性用于样品收集,因为这允许完全回收掺入的血管紧张素-(1-12)。当在37°C下孵育不稳定的血浆时,在人类血液样品中无法检测到血管紧张素-(1-12),而加入到不稳定的人血浆中的血管紧张素-(1-12)在10分钟内消失。稳定的人体血液样本含有血管紧张素II,而血管紧张素-(1-12)检测不到。血,心,和肾脏,但不是大脑,野生型小鼠和大鼠含有可检测水平的血管紧张素II,而血管紧张素-(1-12)检测不到。在肾素敲除小鼠中,所有的血管紧张素,包括血管紧张素-(1-12),在所有地点都无法检测到,尽管血管紧张素原增加了50%.人血浆中的血管紧张素-(1-12)代谢不受肾素抑制的影响。然而,阻断血管紧张素转换酶和氨肽酶A,但不是糜蛋白酶,中性内肽酶,或者脯氨酸寡肽酶,延长血管紧张素-(1-12)的半衰期,血管紧张素转换酶的抑制阻止了血管紧张素II的形成。
■我们无法在人类或小鼠中检测到完整的血管紧张素-(1-12),无论是血液还是组织,这表明这种代谢物不太可能是内源性血管紧张素的来源。
UNASSIGNED: Angiotensin-(1-12), measured by a self-developed, polyclonal antibody-based radioimmunoassay, has been suggested to act as an alternative precursor of angiotensin II. A more reliable detection method would be liquid chromatography-tandem mass spectrometry.
UNASSIGNED: We set up the quantification of human and murine angiotensin-(1-12) by liquid chromatography-tandem mass spectrometry and then used this method to measure angiotensin-(1-12) in human and mouse blood samples, as well as in mouse brain and kidney. We also verified ex vivo angiotensin-(1-12) generation and metabolism in human blood samples incubated at 37 °C.
UNASSIGNED: Stabilization of blood in guanidine hydrochloride was chosen for sample collection since this allowed full recovery of spiked angiotensin-(1-12). Angiotensin-(1-12) was undetectable in human blood samples when incubating nonstabilized plasma at 37 °C, while angiotensin-(1-12) added to nonstabilized human plasma disappeared within 10 minutes. Stabilized human blood samples contained angiotensin II, while angiotensin-(1-12) was undetectable. Blood, hearts, and kidneys, but not brains, of wild-type mice and rats contained detectable levels of angiotensin II, while angiotensin-(1-12) was undetectable. In renin knockout mice, all angiotensins, including angiotensin-(1-12), were undetectable at all sites, despite a 50% rise in
angiotensinogen. Angiotensin-(1-12) metabolism in human blood plasma was not affected by renin inhibition. Yet, blockade of angiotensin-converting enzyme and aminopeptidase A, but not of chymase, neutral endopeptidase, or prolyl oligopeptidase, prolonged the half-life of angiotensin-(1-12), and angiotensin-converting enzyme inhibition prevented the formation of angiotensin II.
UNASSIGNED: We were unable to detect intact angiotensin-(1-12) in humans or mice, either in blood or tissue, suggesting that this metabolite is an unlikely source of endogenous angiotensins.