Walter (Sunny) Dzik, MD, Blood Transfusion Service, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA E-mail: email@example.com
Three vital gases – oxygen, carbon dioxide and nitrogen – intersect at the level of the human red blood cell. The delivery of oxygen to all tissues by red cells is essential to human life. Evolution has created a complex molecule, haemoglobin, designed for efficient uptake and off-loading of oxygen. Iron rests at the centre of the haeme moiety and is critical for oxygen exchange. Although studied for over a century, some details of oxygen transport by the red cell remain uncertain. Recent research has focused on the interaction of haemoglobin with a complex of cell-membrane proteins centred on band 3. In addition, there is renewed interest in the question of whether or not stored red cells deliver oxygen to tissues as well as fresh red cells. The interactions of CO2 with the red cell have drawn far less research attention. Plasma CO2 released by tissues serves as an essential trigger for oxygen release by haemoglobin via the Bohr effect. CO2 transport depends on conversion of CO2 to bicarbonate via red cell carbonic anhydrase in conjunction with chloride exchange across the red cell membrane. There is very little research on the effect of blood storage on CO2 excretion although this aspect of respiratory physiology is every bit as important as oxygen delivery. Nitric oxide (NO) physiology is directly related to oxygen delivery by the red cell. NO serves a local vasodilator to increase blood flow to hypoxic tissue beds. NO binds strongly to haemoglobin which serves as an NO sink. Under normal conditions, plasma NO synthesized by endothelial cells is not consumed by red cell haemoglobin because of a diffusion blockade across the red cell membrane that results from membrane structures not fully identified. Under conditions of red cell lysis, free haemoglobin scavenges NO reducing local vasodilation. Scavenging of NO is now recognized as an important component of the physiologic response to chronic haemolysis and is very likely to play an important role in the renal lesion of acute haemolysis. The three RBC gases are also the three principal gases of the Earth’s atmosphere. While CO2 is the least abundant of the three by far, it is also likely to be the most critical to the future survival of life on Earth – because small further increases in the concentration of CO2 will result in continued climate change, and large increases will be deadly. Thus, the management of three vital gases by the collection of red cells found within us has broad similarities to the collective management of our atmosphere. Just as the survival of individual tissue cells depends upon proper balance of these three respiratory gases, so too will their proper balance be the key to survival of life on Earth.
The reversible binding of oxygen to haemoglobin is an event vital to the life of many species including humans. Oxygen binds co-operatively to haemoglobin such that oxygenation of one globin chain facilitates oxygenation of the other chains in the haemoglobin tetramer. At the centre of haemoglobin is a molecule of iron – held perpendicular to the plane of carbons in the porphyrin ring of haeme. In the capillaries, interaction of haemoglobin with CO2, H+ and Cl− triggers the release of oxygen. Similar reactions, run in reverse, result in uptake of oxygen at the alveolar-capillary interface of the lung. At the maternal–foetal capillary interface, the gradient of O2 is not as profound as that found in the lung. In this unique setting, molecular 2,3 DPG diphosphoglycerate plays a vital role to enhance transfer of O2 from maternal to foetal blood. Foetal haemoglobin, lacking β-globin chains cannot bind 2,3 DPG, and thus more avidly binds oxygen.
Binding of O2 to haeme iron (Fe2+) modifies the charge of iron pulling Fe2+ closer to a histidine residue of globin, and thus changing the overall shape of haemoglobin. With sequential loading and discharge of O2, the haemoglobin molecule alternates between a tense and relaxed state. This cycle repeats itself countless times throughout our lives.
The red cell cytoskeleton is an example of a tensegrity structure. Other examples include Buckminster Fuller’s geodesic dome. This property gives the red cell volume, plasticity and inherent pore sizes. Recent studies show that deoxy-haemoglobin binds to the amino terminus of Band 3 at a location adjacent to key cytoskeletal proteins [1,2]. 2,3 DPG also binds to the red cell cytoskeleton where its effect is to loosen the linkages between cytoskeletal ankyrin, Band 3 and protein 4.1. Taken together, the physical connection between 2,3 DPG, deoxy-haemoglobin and the red cell cytoskeleton suggests that changes in the oxygenation status of haemoglobin may be co-ordinated with changes in the red cell membrane. This observation may be directly relevant to loss of red cell plasticity during blood storage and may account, in part, for unique interactions of the red cell with nitric oxide (see below) .
Clinical studies of O2 delivery by red cells
Increasing the delivery of oxygen to tissues is the primary indication for the tens of millions of units of blood transfused annually. Nevertheless, we have limited ways to monitor tissue oxygenation and measure the effect of transfusion. New techniques, under evaluation, may ultimately provide better information on the effect of transfusion [3–5]. Near infrared spectroscopy has been applied to monitor cerebral circulation (INVOS system: http://www.somanetics.com/invos.asp). As shown in Fig. 1, a non-invasive sensor is placed on the forehead, and the device measures real-time changes in regional oxygen saturation of the brain. Light, emitted from a source applied to the scalp, is measured by a detector at two depths: surface light (trans-illumination) and deeper light (2·5–3 cm) reflected by underlying brain tissue. After accounting for surface light, the amount and wavelength of reflected deeper light is used to measure the proportion of total haemoglobin that is de-oxygenated. A similar device (InSpectra™, http://www.htibiomeasurement.com) has also been developed for use on the thenar muscle of the hand as a measure of peripheral tissue oxygenation. A study, conducted in trauma patients, used this device to generate preliminary data on the oxygenation of tissue following transfusion . Another new technique is designed to observe microvascular blood flow. This technology, termed side-stream dark field (SDF) microscopy, illuminates mucosal tissue with 530 -nm green light. Haemoglobin absorbs light at this wavelength, and as a result, the flowing blood appears as dark images against a grey background. A commercially available device exists (Microscan™) .
In sub-Saharan Africa, severe anaemia as a consequence of malaria represents the most common indication for transfusion, accounting for up to 80% of blood use. Children with Plasmodium falciparum malaria may develop haemoglobin values < 50 g/l accompanied by lactic acidosis. For those with profound anaemia (Hg < 30 g/l), the transfusion of red blood cells is a life-saving medical miracle. The extreme anaemia of malaria which is not complicated by heart disease, sepsis, hypovolemia or shock represents an opportunity to study the physiology of tissue oxygenation in response to blood transfusion .
The efficient removal of CO2 from tissues is as vitally important as the delivery of oxygen . In the capillary bed, the concentration of CO2 is the principal trigger for the release of oxygen by erythrocytes. Three steps occur: CO2 transport into the red cell, conversion by carbonic anhydrase and the exchange of bicarbonate for chloride . First, CO2 enters the red cell where it encounters red cell carbonic anhydrase located just beneath the membrane surface. Carbonic anhydrase is a key enzyme in human physiology. It converts CO2 to bicarbonate and a hydrogen ion as:
The resulting intracellular is then exchanged for extracellular Cl− by the red cell membrane anion exchanger. This leads to an increase in intracellular H+ and extracellular . The net effect is for the carbon of CO2 to be exported from the red cell and carried in the plasma as bicarbonate. Meanwhile, the intracellular H+ binds to haemoglobin and serves as a primary signal to release oxygen, a phenomenon known as the Bohr effect. The above mechanism accounts for approximately 60% of the removal of CO2. An additional 20% is carried by direct binding to haemoglobin, giving venous blood a bluish tint. And approximately 20% is carried as dissolved CO2 in the plasma accounting for the elevated partial pressure of CO2 in venous blood.
Clinical studies of CO2 removal by red cells
Few studies have examined the direct effect of transfusion on CO2 physiology. Several areas of research opportunity exist. These include the effect of refrigerated blood storage on red cell carbonic anhydrase function or on the capacity for bicarbonate/chloride exchange. Clinical studies, designed to investigate the effect of transfusion, might consider a focus on clearance of CO2 as a result of transfusion, rather than on the delivery of oxygen. For some diseases characterized by high CO2 production or impaired CO2 clearance, transfusion might be viewed as means to enhance CO2 removal.
Under normal physiologic circumstances, NO is a vasodilator of deoxygenated tissue beds. This effect, termed ‘hypoxic vasodilation,’ increases blood flow to poorly oxygenated tissues . In addition, NO inhibits platelet function and may serve as a normal vascular anti-thrombotic agent. NO may be especially relevant at the level of arterioles and venules where the speed of blood flow is decreased. Because the pulmonary artery vasculature is deoxygenated, the low pressure found in the pulmonary vasculature may depend in part from the effects of NO. In conditions where NO is depleted, such as sickle cell disease, the pulmonary circuit may be particularly susceptible with resulting pulmonary hypertension .
The current prevailing view is that the red cell takes up NO and releases it under conditions of hypoxia. Extensive research has documented that NO binds to oxy-haemoglobin and is either directly released by deoxy-haemoglobin  or is release via the action of nitrite reductase . While the uptake and release of NO by haemoglobin is irrefutable, the quantity of NO delivered by haemoglobin-transport remains difficult to measure. A most interesting alternative hypothesis is based on the idea that the red cell membrane may demonstrate a difference in permeability to NO depending on the oxygen status of haemoglobin . See Fig. 2. This hypothesis suggests that when haemoglobin is oxygenated, the red cell membrane is permeable to NO. As a result, NO produced by endothelial cells passes through the red cell membrane and is absorbed by haemoglobin. However, when haemoglobin is deoxygenated, the red cell membrane may become impermeable to NO. Thus, in hypoxic vascular beds, the NO synthesized by endothelial cells is not captured by haemoglobin and is available to exert a local vaso dilatory effect on vascular smooth muscle. The end result achieves the characteristic hypoxic vasodilation mediated by NO.
The importance of the red cell membrane in NO physiology is demonstrated by clinical conditions characterized by intravascular haemolysis or deliberate infusion of stroma-free haemoglobin [15,16]. In these states, in which the effect of the red cell membrane is absent, NO released from endothelial cells is absorbed by plasma-free haemoglobin. The depletion of NO by plasma haemoglobin is referred to as ‘NO scavenging’ and produces a clinical picture characterized by renal vasoconstriction, hypertension, platelet activation, WBC adhesion and RBC rigidification. For phenomena related to NO physiology, the striking clinical contrast between conditions when haemoglobin is properly encapsulated with the cell membrane compared with conditions when haemoglobin is released into the plasma suggest that simple uptake and release of NO by haemoglobin does not fully explain NO physiology. Instead, the observed clinical consequences of haemolysis suggest an important role for the red cell membrane in the normal control of NO physiology.
The vital gases and Earth’s atmosphere: a primer on global climate change
The three most important gases carried by the human red cell are also the three most plentiful gases in the Earth’s atmosphere: Nitrogen (78%), Oxygen (21%) and CO2 (0·038%). Although CO2 occupies by far the smallest proportion of the atmosphere, its relative concentration is changing the most quickly and rising CO2 levels are expected to have dramatic effects on the Earth’s climate. Apart from water vapour, CO2 is the most plentiful of the greenhouse gases. Greenhouse gases affect the balance of solar energy which remains on Earth. Solar energy, arriving as light, is of short wavelength and easily penetrates through the greenhouse gas layer. The Earth then reflects an enormous amount of solar energy back into space in the form of long-wavelength radiant heat. Greenhouse gases block a portion of that outflow of energy, trapping solar heat, on the planet.
In recent decades, there has been a dramatic rise in the concentration of CO2 in the atmosphere as a result of an enormous increase in the burning of fossil fuels by industrialized nations. The amount of carbon released into the Earth’s atmosphere, as measured in millions of metric tonnes per year, has been increasing steadily for 50 years.
The concentration of CO2 in the atmosphere is a basic indicator of the extent of fossil fuel combustion and its impact on the Earth’s atmosphere. Two features are worthy of note. First, as shown from direct measurement at Mauna Loa, Hawaii, the concentration of CO2 has risen each year during the last half-century. Secondly, levels of CO2 have been measured in air bubbles trapped within ice found deep within the Antarctic ice shelf. The depth of each ice sample corresponds to the age of the ice. This has provided a record of the CO2 content in air dating back hundreds of thousands of year. See Fig. 3. As shown in the figure, there is a natural periodicity to the concentration of CO2 over the millennia with levels fluctuating between 200 to 300 ppm every 100 000 years. The consistency of this periodicity is striking as is the fact that the highest levels never exceed 300 ppm – except until recently, where levels now approach 400 ppm. These data demonstrate that the Earth’s atmosphere has now reached a CO2 level not witnessed at any previous time. Coupled with the information on carbon release into the atmosphere, it is very difficult to claim that the current high levels of CO2 are the result of a natural planetary cycle. Instead, the evidence argues persuasively that we have substantially disturbed the natural cycle of our planet.
The consequences of rising CO2 in the Earth’s atmosphere
Because CO2 is a greenhouse gas, the principal consequence of its rising concentration is a decrease in escape of heat reflected from the Earth. The rise in CO2 concentration has been accompanied by five parallel changes in the Earth’s climate: First, the average global temperature has risen 1·5°C since 1880.
While the absolute rise in temperature may not seem dramatic, it has been observed that only 5°C separates our current average global temperature from that seen during the ice age. Secondly, serial satellite images document that the northern polar ice cap is melting. The United States National Aeronautics and Space Administration estimates that northern polar ice melt amounts to approximately 300 000 square miles in the last decade. An important consequence of this melting will be loss of the polar ice cap’s capacity to reflect incoming solar energy, thus further accelerating the warming of the planet. Thirdly, photographs, taken from the same vantage point a decade or more apart, document dramatic shrinkage of glaciers in Europe, North America, South America and Oceania. Fourthly, as increasing amounts of CO2 are dissolved into the ocean, the pH of the oceans has decreased. Serial measurements document a decline in ocean pH from 8·11 to 8·08 over the past 18 years. Fifthly, serial satellite images also document a dramatic loss of ice from Greenland (Fig. 4). Unlike the floating ice of the arctic polar cap, the ice of Greenland is on land, and its melting adds new water to the world’s oceans. The melting of Greenland’s ice will raise the sea level of the planet. If the present course of melting continues, the rise in sea level would have dramatic consequences for large population centres on Earth.
Three vital respiratory gases – oxygen, nitrogen and carbon dioxide – intersect at the level of the human red cell. The physiology of these gases is interrelated and depends upon important interactions with the red cell membrane. New techniques, both research and clinical, may offer new insights into the indications for transfusion and treatment of haemolytic disorders. Ultimately, the three vital gases that determine the fate of individual tissue cells are the same three that compose our atmosphere and upon which our collective fate depends.