Respiratory Substrates and the Respirometer

Spec Mapping — OCR H420 Module 5.2.2 — Respiration, content statements covering respiratory substrates (carbohydrates, lipids, proteins) and their relative energy yields, the calculation and interpretation of respiratory quotient (RQ), and the use of a respirometer to measure rate of respiration in small organisms and tissues (refer to the official OCR H420 specification document for exact wording).

Glucose is the classic respiratory substrate, but in reality cells can respire lipids, proteins and other carbohydrates too. Each substrate yields a different amount of ATP and a different ratio of CO₂ produced to O₂ consumed — the respiratory quotient (RQ). OCR specification module 5.2.2 requires you to know the main substrates, their relative energy content, the concept of RQ and how to measure respiration rate and RQ using a respirometer. This lesson combines biochemistry with a key OCR practical.

The respirometer itself has a distinguished pedigree. Otto Warburg (Berlin, 1920s) developed the Warburg manometer, a constant-volume gas-pressure apparatus that allowed tissue slices, microbial cultures and isolated mitochondria to be studied quantitatively. Paraphrasing Warburg's school of thought, the rate of oxygen consumption was the master variable of cellular bioenergetics — visible directly through gas-volume change. Warburg's manometric work led to the discovery of the cytochrome respiratory enzymes (for which he received the 1931 Nobel Prize) and to his still-cited observation that tumour cells preferentially use glycolysis even in the presence of oxygen (the "Warburg effect"). Modern respirometry has shifted from manometers to Clark-type oxygen electrodes and computerised gas-exchange systems, but the underlying principle — measuring O₂ consumption and CO₂ production with CO₂-absorbers and control tubes — is essentially unchanged.

Key Definitions:

• Respiratory substrate — any organic molecule that can be oxidised by cells to release energy.
• Respiratory quotient (RQ) — the ratio of CO₂ produced to O₂ consumed during respiration (RQ = CO₂/O₂).
• β-oxidation — the pathway that breaks fatty acids into 2-carbon fragments (acetyl CoA) for entry into the Krebs cycle.
• Deamination — the removal of an amino group from an amino acid, producing urea and a keto acid.
• Respirometer — a piece of apparatus used to measure the rate of oxygen consumption by a small organism or sample of tissue.

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The Three Main Substrates

1. Carbohydrates

• Primarily glucose, but also starch and glycogen after hydrolysis.
• Enters directly into glycolysis.
• Yield: ~32 ATP per glucose (or equivalent per monosaccharide).
• Energy density: ~17 kJ g⁻¹.
• First choice fuel in most tissues because it is easy to mobilise and enters respiration directly.

2. Lipids (Triglycerides)

• Hydrolysed to glycerol + 3 fatty acids by lipases.
• Glycerol is converted to TP and enters glycolysis.
• Fatty acids enter the mitochondrial matrix and undergo β-oxidation: they are chopped two carbons at a time into acetyl CoA molecules, which enter the Krebs cycle directly.
• β-oxidation itself also reduces NAD and FAD, adding to the ETC input.
• Energy density: ~39 kJ g⁻¹ — more than double that of carbohydrates!
• This high energy density is why fat is the body's long-term energy store.

Why Do Lipids Yield So Much Energy?

Fatty acids are highly reduced (lots of C–H bonds, few C=O bonds). Oxidation releases a lot of energy — each C-H bond is a rich source of electrons for the ETC. A typical 18-carbon fatty acid (stearate) yields roughly 120 ATP through β-oxidation and the Krebs cycle, compared to ~32 for a single 6-carbon glucose.

β-Oxidation in Outline

flowchart LR
    FA[Fatty acid - CoA] --> B1[Remove 2C as acetyl CoA]
    B1 -->|Reduce NAD and FAD| KR[Krebs cycle]
    B1 --> FA2[Shortened fatty acid]
    FA2 --> B1
    KR --> ETC[ETC -> ATP]

Each round of β-oxidation removes a 2-carbon unit, producing: 1 acetyl CoA + 1 reduced NAD + 1 reduced FAD. A long fatty acid goes round many times.

3. Proteins

• Used as a respiratory substrate only when carbohydrates and lipids are exhausted, e.g. during starvation or extreme exercise.
• Hydrolysed to amino acids by proteases.
• Amino acids are deaminated in the liver (the amino group is removed as NH₃, then detoxified to urea via the ornithine cycle and excreted).
• The remaining keto acids enter the Krebs cycle at various points depending on the amino acid (e.g. alanine → pyruvate, glutamate → α-ketoglutarate, aspartate → oxaloacetate).
• Energy density: ~17 kJ g⁻¹, similar to carbohydrates.

Using proteins as fuel has a cost: it depletes the body's own muscle and enzyme proteins, and the excretion of urea requires water and ATP. It is a last resort.

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Comparing the Substrates

Substrate	Energy density (kJ/g)	ATP per glucose equivalent	Typical use
Glucose	~17	~32	Default, immediate energy
Lipids (stearic acid)	~39	~120 per C₁₈ fatty acid	Long-term storage, endurance
Proteins (amino acids)	~17	Variable; ~15–30 per amino acid	Starvation, severe stress

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Respiratory Quotient (RQ)

RQ is defined as:

$RQ = \frac{\text{volume of CO}_2 \text{ produced}}{\text{volume of O}_2 \text{ consumed}}$RQ=volume of O2​ consumedvolume of CO2​ produced​

The RQ depends on the chemical composition of the substrate — specifically, how many hydrogens it has per carbon, because more hydrogens means more oxygen is needed relative to the carbon content.

Typical RQ Values

Substrate	Equation	RQ
Carbohydrate	C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O	1.0
Lipid (e.g. triolein)	C₅₇H₁₀₄O₆ + 80O₂ → 57CO₂ + 52H₂O	~0.7
Protein	Variable (amino acids)	~0.9
Mixed diet	—	~0.85

Interpreting an RQ

• RQ = 1.0 → pure carbohydrate respiration.
• RQ = 0.7 → pure lipid respiration.
• RQ = 0.85 → mixed substrate (typical human diet).
• RQ > 1.0 → anaerobic respiration may be contributing (extra CO₂ released without corresponding O₂ uptake).
• RQ < 0.7 → unusual; may indicate fixation of CO₂ (e.g. some plants in light conditions) or conversion of carbohydrate to lipid (lipogenesis).

A Worked Example

If a cell consumes 20 cm³ of O₂ and produces 15 cm³ of CO₂, then:

• RQ = 15 / 20 = 0.75
• This is close to the lipid value, suggesting mainly fat is being respired.

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The Respirometer: Required Practical

OCR practical activity group 12 requires you to use a respirometer to measure the rate of respiration of small organisms (e.g. germinating seeds, maggots, woodlice) or cell samples.

The Apparatus

flowchart LR
    O[Organism] --> T1[Test tube with KOH]
    O -.CO2 absorbed.-> KOH[Potassium hydroxide solution]
    T1 --> CT[Capillary tube with coloured manometer fluid]
    CT --> C[Control tube - no organism - compensates for pressure/temperature changes]
    WB[Thermostatic water bath]

Principle

• Place the organism in a sealed tube containing a CO₂ absorber (KOH or soda lime) so that any CO₂ released is immediately absorbed.
• As the organism respires, it consumes O₂ — this reduces the gas volume in the tube.
• The reduction in volume causes the coloured fluid in the capillary manometer to move towards the organism.
• Use a scale to measure how far the fluid has moved.
• The volume of O₂ consumed per unit time is the rate of respiration.

Why Use KOH or Soda Lime?

• CO₂ is produced by the organism but must be absorbed so that it does not cancel out the decrease in gas volume from O₂ consumption.
• KOH + CO₂ → K₂CO₃ + H₂O (or equivalent with soda lime).
• If KOH were not present, the CO₂ would replace some of the O₂ in the gas volume, and the fluid would not move as much — you would underestimate the rate of respiration.

Controls

• A control tube with glass beads (instead of an organism) is set up identically. This controls for changes in pressure or temperature not caused by respiration.
• The water bath keeps the temperature constant (typically 20°C or 25°C).
• Equilibrate for ~5 minutes before starting measurements.

Calculating the Rate

If the fluid moves by distance d in the capillary (in mm), and the capillary has cross-sectional area A (in mm²), then:

$\text{Volume of O}_2 \text{ consumed} = A \times d$Volume of O2​ consumed=A×d

Rate = volume ÷ time. Units: cm³ O₂ per minute per gram of tissue.

Measuring RQ with a Respirometer

To measure RQ, run the experiment twice:

1. First run (with KOH): measures O₂ consumption only. The manometer fluid moves because O₂ is consumed and CO₂ is absorbed.
2. Second run (without KOH): CO₂ is not absorbed, so the net change in gas volume = O₂ consumed − CO₂ produced.
3. Subtract one result from the other to get CO₂ produced.
4. Calculate RQ = CO₂ / O₂.

Variables

• IV: temperature, substrate type, age of organism.
• DV: volume of O₂ consumed per unit time.
• Controlled: mass/number of organisms, species, temperature, pressure, light.

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Factors Affecting Respiration Rate in Living Organisms

• Temperature — higher temperature = faster rate, up to an optimum, after which enzymes denature.
• Activity level — exercise or locomotion increases ATP demand, raising respiration rate.
• Availability of substrate — starved organisms respire less.
• Availability of oxygen — anaerobic conditions slow the rate dramatically and also alter the RQ.
• Life stage — germinating seeds and actively growing tissues respire faster than dormant or quiescent cells.
• Species and metabolic rate — small mammals (e.g. shrews) have very high mass-specific respiration rates.

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Exam Tip

OCR often asks you to calculate a rate of respiration from respirometer data. Always include units (cm³ O₂ min⁻¹ g⁻¹ is the standard) and show your working. If you are given the diameter of the capillary, remember to use A = πr² to find the cross-sectional area, not the diameter. A common trap is forgetting to halve the diameter.

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Common Exam Mistakes

• Forgetting the role of KOH. It absorbs CO₂ so that the only volume change comes from O₂ consumption.
• Confusing "manometer" and "capillary tube". The manometer is the whole U-shape or curved tube; the capillary is the narrow part where fluid movement is measured.
• Saying proteins are the "preferred" fuel. They are a last-resort fuel, used only in starvation.
• Claiming lipids have a lower energy density than carbohydrates. They have more than double (~39 kJ/g vs ~17 kJ/g).
• Writing "RQ is always 1". Only for pure carbohydrate respiration.
• Forgetting that RQ > 1 can indicate anaerobic respiration contributing. Extra CO₂ comes from ethanol fermentation without O₂ being consumed.
• Not subtracting the control reading. Any pressure/temperature changes not due to respiration must be subtracted.
• Writing "RQ has units of cm³". RQ is a ratio and has no units.

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Substrate Comparison — Full Markdown Table

Substrate	Energy density (kJ/g)	Entry into respiration	ATP yield	RQ	Storage form	Mobilisation
Carbohydrate (glucose)	~17	Direct (glycolysis)	~32 ATP per glucose	~1.0	Glycogen (liver, muscle)	Glycogenolysis by phosphorylase
Lipid (triolein C₅₇H₁₀₄O₆)	~39	β-oxidation → acetyl-CoA → Krebs	~120 ATP per C₁₈ fatty acid; ~460 ATP per triglyceride	~0.7	Triglyceride (adipose)	Lipolysis by lipase
Protein (varies)	~17	Deamination → keto acid → Krebs intermediates	~15–30 ATP per amino acid	~0.9	None (functional protein)	Proteolysis (only under starvation)

The ~2.3× energy density of lipids relative to carbohydrates reflects their highly reduced chemical state (long C-H-rich chains, few C-O bonds). More reductions per substrate molecule means more NADH/FADH₂ delivered to the ETC, which means more ATP. Per gram of stored fuel, triglyceride is the body's most efficient long-term energy store — which is why hibernating mammals, migrating birds and oil-secreting seeds rely on lipid stores rather than carbohydrate stores.

KaTeX for RQ Calculations

For glucose: $\text{C}_6\text{H}_{12}\text{O}_6 + 6\,\text{O}_2 \rightarrow 6\,\text{CO}_2 + 6\,\text{H}_2\text{O}$C6​H12​O6​+6O2​→6CO2​+6H2​O

$RQ_{\text{glucose}} = \frac{6\,\text{CO}_2}{6\,\text{O}_2} = 1.0$RQglucose​=6O2​6CO2​​=1.0

For triolein (a representative triglyceride): $\text{C}_{57}\text{H}_{104}\text{O}_6 + 80\,\text{O}_2 \rightarrow 57\,\text{CO}_2 + 52\,\text{H}_2\text{O}$C57​H104​O6​+80O2​→57CO2​+52H2​O

$RQ_{\text{triolein}} = \frac{57}{80} \approx 0.71$RQtriolein​=8057​≈0.71

For protein (variable, average ~0.85–0.90 depending on the amino-acid composition).

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