A respiratory physiology practical class - and beyond

The oxygen has been consumed in the oxidation of metabolic fuels, mainly glucose and fatty acids, in various tissues, and the carbon dioxide is the product of that oxidation.

In the lungs oxygen dissolves in the surface layer of fluid, then diffuses across the epithelial cells (it is lipid soluble) and dissolves in blood plasma. It diffuses into the red blood cells, and is bound by the protein haemoglobin, which has a high affinity for oxygen. This means that there is a continual downwards concentration gradient for oxygen from the alveoli of the lungs to the haemoglobin in red blood cells, although there is only a very small amount of oxygen in solution in plasma at any one time.

Although almost all of the oxygen in the bloodstream is bound to haemoglobin in red blood cells, there is always a small amount in free solution that is available to diffuse into tissues. In muscle the protein myoglobin has a higher affinity for oxygen than does haemoglobin. This ensures a downwards concentration of gradient from red blood cells into muscle, where it is stored bound to myoglobin. Again there is always some oxygen in free solution in cells. The enzymes in cells that require oxygen have a higher affinity for oxygen than does myoglobin, so again, as oxygen is required, it diffuses down a concentration gradient to a protein with a higher affinity.

The total concentration of proteins in blood plasma is approximately 70 g/L, and plasma is noticeably more viscous than water. Adding an additional 140 g/L of protein from haemoglobin in free solution would result in plasma being too viscous to flow adequately.

By packing haemoglobin into red blood cells at a high concentration it is possible to maintain an adequately low viscosity for blood to flow. Although red blood cells are conventionally shown as rigid discs, as in the diagram above, in fact they are very flexible. The high concentration of haemoglobin means that they have a "jelly-like" consistency, enabling them to deform to travel through small blood capillaries, as shown in the picture on the left.

In the genetic disease sickle cell anaemia, the abnormal haemoglobin becomes insoluble and crystallises at low partial pressures of oxygen. When this occurs, red blood cells lose their flexibility and cannot deform to travel through small blood capillaries, so causing a blockage, increasing crystallisation of haemoglobin and a very painful "sickling crisis".

• removal of hydrogen
• removal of electrons
• addition of oxygen

By removal of hydrogen onto an intermediate hydrogen carrier (or coenzyme), either NAD (nicotinamide adenine dinucleotide) or a flavin. See the exercise on overheating after overdosing on E later on for more on these coenzymes.

During the practical class it was also noticed that the exhaled air contained considerably more water vapour than inhaled air. Just over 1 mL more water was collected from exhaled air per minute than was present in the air in the laboratory.

This led to a separate experiment in which PN and several friends measured their intake of fluids, the water content of their food and the volume of urine passed each day for three days.

You could "cheat" by weighing the foods eaten and using food composition tables, but a more precise method would be to weight each food served, then take a small weighed sample and dry it to constant weight in an oven at 100°C. The difference in weight is due to material that is volatile at 100°C - i.e. water.

A metabolic chamber is a sealed room. There is an inlet for air from the laboratory, and air is pumped out of the chamber at a constant known through a condenser (which condenses water vapour to liquid water, which can then be measured). It is easy to measure the water content of the air in the laboratory by similarly using a condenser through which air is pumped at a constant known rate.

The air entering the chamber, and that leaving can also be sampled for measurement of oxygen an carbon dioxide, and heat output from the body can be measured by circulating cold water through pipes in the walls at such a rate as to maintain a constant temperature in the chamber. The increase in temperature of this cooling water then allows calculation of heat output in the chamber. You will come across use of such metabolic chambers to measure energy expenditure in later exercises.

Rather than collect their faeces to determine the water content in this experiment, they used a literature average figure - an adult man loses about 100 mL of water in faeces each day.

At the end of the experiment they drew up a table of their average daily fluid balance - the results were as follows:

The end products of oxidation of metabolic fuels (mainly fatty acids and glucose) are carbon dioxide and water. This is what is known as metabolic water - water produced by the oxidation of metabolic fuels.

for glucose:
C6H12O6 + 6 O2 = 6 CO2 + 6 H2O

for palmitate:
C15H31COOH+ 23 O2 = 16 CO2 + 16 H2O

At this stage PN visits a friend who is studying for a PhD in the Department of Medicine. Her project involves the control of metabolism in isolated liver cells from normal and diabetic rats.

In one experiment she incubated the cells in stoppered vessels with an atmosphere containing the stable isotope of oxygen, 18O.

The cells were incubated in the outer compartment of the flask. At the end of the incubation an alkaline solution was injected through the stopper into the centre well, and a non-volatile acid (trichloroacetic acid) was injected into the outer compartment, to stop the reaction and drive off carbon dioxide, which was absorbed by the alkali in the centre well.The water in the incubation mixture was then micro-distilled the water into a separate vessel. This allows measurement of 18O in both carbon dioxide and water.

In one set of incubations the cells were provided with glucose as their substrate, and in the other with palmitate

If there is no 18O label in the carbon dioxide produced by the metabolism of glucose, and label only appears in water, then the oxidation of glucose and palmitate to carbon dioxide must occur only by removal of hydrogen. The oxygen that has been consumed must all have been used to oxidise that hydrogen to water.

The only other possibility is water. Oxygen can be introduced into a compound by forming a carbon-carbon double bond by removal of hydrogen, followed by the addition of water across this double bond to form a hydroxyl group. The reactions shown below are examples these two types of oxidation

The obvious experiment would be to incubate the cells with palmitate or glucose in the presence of 18O labelled water. You would indeed then see label in the carbon dioxide.

The reactions shown above represent a simple metabolic pathway - a series of chemical reactions, each catalysed by a different enzyme, with generally only one small change in the substrate at each step. By linking a number of enzyme-catalysed reactions in a sequence or pathway, where the product of one enzyme-catalysed reaction is the substrate for the next, it is possible to perform a complex chemical change in a series of simple steps.

Equally, a chemical reaction such as the oxidation of ethanol to carbon dioxide and water, which occurs rapidly in vitro, simply by setting light to the ethanol in air, with no detectable intermediates, proceeds via 11 separate enzyme-catalysed steps in vivo, and also requires the mitochondrial electron transport chain.

You can ensure a constant unidirectional flux through a pathway in two ways:

a) by providing a constant source of the initial substrate
b) more commonly, by removing one or more of the products of the pathway as a whole or of one or more individual reactions

In the example shown here the pathway proceeds to the total oxidation of ethanol because the carbon dioxide formed is lost from the cell, and eventually lost from the body in exhaled air. Just as there is a continual downwards concentration gradient of oxygen from the lungs (where it is inhaled) to the tissues (where it is used), so there is a continual downhill gradient of carbon dioxide from tissues (where it is produced) to the lungs, where it is exhaled.

Key points from this exercise:

• Oxygen is consumed in the oxidation of metabolic fuels to carbon dioxide and water.
• Oxidation can occur by:
  • removal of hydrogen (hydrogen ions plus electrons)
  • removal of electrons
  • addition of oxygen from oxygen itself or from water.
• Most metabolites are oxidised by removal of hydrogen onto an intermediate hydrogen carrier (a coenzyme).
• The water formed by the oxidation of metabolic fuels is known as metabolic water. Total water loss from the body is about 350 mL greater than water intake.
• A metabolic pathway is a series of enzymes catalysing reactions, in which the product of one enzyme acts as the substrate of the next.
• Unidirectional flux through a pathway, even when all the enzymes are readily reversible, can be ensured by providing a constant source of the starting substrate, or, more commonly, by removing one or more of the products of the pathway.