Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy
Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy
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DOI：
10.1098/rstb.1997.0048
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发表时间：
1997-06-29
影响因子：
6.3
通讯作者：
Springett, R
中科院分区：
文献类型：
--
作者：
Cooper, CE;Springett, R
细胞色素氧化酶是线粒体呼吸链的末端电子受体。它负责体内绝大多数的氧气消耗，并对细胞ATP的有效生成至关重要。该酶含有四个氧化还原活性金属中心;其中之一，双核Cu-A中心，在近红外线中具有强吸收，使其能够通过近红外光谱在体内检测。然而，该中心的浓度小于血红蛋白的chat的10%这一事实意味着它的检测不是一件小事。与脱氧血红蛋白和氧合血红蛋白的情况不同，总细胞色素氧化酶蛋白的浓度变化非常缓慢（几天），因此不容易通过近红外光谱检测。然而，铜中心迅速接受并提供电子，因此可以迅速改变其氧化还原状态;这种氧化还原变化可以通过近红外光谱检测。在体内，许多因素都会影响铜-A的氧化还原状态（库珀等人，1994年），但最重要的可能是分子氧浓度（在低氧张力下，随着酶对氧的还原开始限制电子转移的稳态速率，电子在Cu-A上积累）。血红蛋白氧合、脱氧和血容量变化的潜在因素通常是，进行近红外光谱测量的临床医生和生理学家很好地理解。相反，控制细胞色素氧化酶中铜-A的稳态氧化还原水平的因素仍然是一个活跃的争论问题，甚至在研究分离的酶和线粒体的生物化学家之间。再加上精确的体内测量的困难，细胞色素氧化酶近红外领域的发展可能并不令人惊讶。光谱学的过去多少有些曲折。很多时候，论文中的信息不足，无法重复测量，而且很少尝试在体内测试算法。近年来，许多研究小组和商业光谱仪制造商进行了一致的尝试，不仅说明他们如何尝试通过近红外光谱法测量细胞色素氧化酶，而且还证明：他们真的在这样做。我们赞赏这些尝试，这些尝试一般分为三个方面：首先，可以进行数据建模，以确定哪些问题可能会破坏细胞色素氧化酶检测算法（Matcher等人，1995）;其次，可通过血液稀释改变血红蛋白浓度（使用生理盐水或人造血液替代品）在动物中（Tamura 1993）或患者（Skov & Greisen 1994）;第三，细胞色素氧化酶的氧化还原状态可以通过使用线粒体抑制剂来固定，显著改变血红蛋白氧合、血红蛋白浓度和光散射（库珀et al. 1997）。我们以前写过综述，涵盖了在体内测量细胞色素氧化酶近红外光谱信号的困难（库珀et al. 1997）和影响细胞色素氧化酶Cu-A氧化态的因素（库珀et al. 1994）。在这篇文章中，我们想打击一个更乐观的注意，我们将强调有用的测量可能在临床环境中，以及描述的条件下，我们可以有信心，我们正在测量真实的变化的铜-A氧化还原状态。
Cytochrome oxidase is the terminal electron acceptor of the mitochondrial respiratory chain. It is responsible for the vast majority of oxygen consumption in the body and essential for the efficient generation of cellular ATP. The enzyme contains four redox active metal centres; one of these, the binuclear Cu-A centre, has a strong absorbance in the near-infrared that enables it to be detectable in vivo by near-infrared spectroscopy. However, the fact that the concentration of this centre is less than 10% of chat of haemoglobin means that its detection is not a trivial matter.Unlike the case with deoxyhaemoglobin and oxyhaemoglobin, concentration changes of the total cytochrome oxidase protein occur very slowly (over days) and are therefore not easily detectable by near-infrared spectroscopy. However, the copper centre rapidly accepts and donates an electron, and can thus change its redox state quickly; this redox change is detectable by near-infrared spectroscopy. Many factors can affect the Cu-A redox state in vivo (Cooper et al. 1994), but the most significant is likely to be the molecular oxygen concentration (at low oxygen tensions, electrons build up on Cu-A as reduction of is oxygen by the enzyme starts to limit the steady-state rate of electron transfer).The factors underlying haemoglobin oxygenation, deoxygenation and blood volume changes are, in general, well understood by the clinicians and physiologists who perform near-infrared spectroscopy measurements. In contrast, the factors that control the steady-state redox level of Cu-A in cytochrome oxidase are still a matter of active debate, even amongst biochemists studying the isolated enzyme and mitochondria. Coupled with the difficulties of accurate in vivo measurements it is perhaps not surprising that the field of cytochrome oxidase near-infrared. spectroscopy has a somewhat chequered past. Too often papers have been written with insufficient information to enable the measurements to be repeated and few attempts have been made to test the algorithms in vivo.In recent years a number of research groups and commercial spectrometer manufacturers have made a concerted attempt to not only say how they are attempting to measure cytochrome oxidase by near infrared spectroscopy but also to demonstrate that the!: are really doing so. We applaud these attempts, which in general fall into three areas: first, modelling of data can be performed to determine what problems are likely to derail cytochrome oxidase detection algorithms (Matcher et al. 1995); secondly haemoglobin concentration changes can be made by haemodilution (using saline or artificial blood substitutes) in animals (Tamura 1993) or patients (Skov & Greisen 1994); and thirdly, the cytochrome oxidase redox state can be fixed by the use of mitochondrial inhibitors and then attempts made to cause spurious cytochrome changes dy dramatically varying haemoglobin oxygenation, haemoglobin concentration and light scattering (Cooper et al. 1997).We have previously written reviews covering the difficulties of measuring the cytochrome oxidase near infrared spectroscopy signal in vivo (Cooper et al. 1997) and the factors affecting the oxidation state of cytochrome oxidase Cu-A (Cooper et al. 1994). In this article we would like to strike a somewhat more optimistic note-we will stress the usefulness this measurement may have in the clinical environment, as well as describing conditions under which we can have confidence that we are measuring real changes in the Cu-A redox state.