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Metabolism can be investigated at different biological levels, each giving a unique window into how pathways function in living systems.
Whole-animal studies reveal integrated physiology.
Key examples:
• Feeding experiments identified essential amino acids and vitamins.
• Benzoic acid → hippuric acid conversion discovered by Wohler (1842).
• Radiolabelled iron (⁵⁹Fe) used to study RBC turnover and heme metabolism.
• Inborn errors of metabolism help uncover normal biochemical pathways.
Organ is removed with intact vessels and perfused with Ringer solution.
• Test substrates added to perfusion fluid
• Outflow analyzed to determine metabolic changes
Ideal for organ-specific metabolism.
Thin slices (~50 µm) preserve internal organelles.
• Introduced by Otto Warburg
• Used for measuring respiration and glucose oxidation
• Liver slices can be used to assess CO₂ evolution
Warburg apparatus measures tissue respiration.
Cells are cultured in a nutrient-rich medium (pH ~7.2).
Used to study:
• Radiolabelled glucose metabolism
• DNA synthesis using labelled nucleotides
• Drug toxicity and drug response
• Biological product synthesis (e.g., monoclonal antibodies)
HeLa cells are classic immortal human cell lines.
Tissues are homogenized and organelles separated.
Used to study:
• Mitochondrial function
• Electron transport chain activity
• Subcellular enzyme function
Isolated enzyme systems help determine:
• Reaction mechanism
• Cofactor requirement
• Regulation of enzyme activity
Modern metabolic research focuses on gene-level study.
• Mutation analysis (e.g., PAH mutation in PKU)
• Genomics – entire set of genes
• Transcriptomics – gene expression
• Proteomics – protein profile patterns
Radiolabelled compounds track metabolism.
Examples:
• ¹⁴C-glucose → traced to metabolic end-products in organs
• ¹⁵N-glycine → incorporated into hemoproteins, nucleic acids, creatinine
Tracing helps identify:
• Metabolic pathways
• Precursor–product relationships
• Turnover rates
• Organ-specific metabolism
• Metabolism is studied at six levels: whole organism → perfused organ → organ slices → intact cells → tissue homogenates → purified enzymes/DNA.
• Warburg apparatus measures oxygen consumption.
• Tissue culture allows drug testing, metabolic flux studies, DNA synthesis studies.
• Radioisotopes map metabolic pathways and turnover.
• HeLa cells are immortal human cancer cells widely used in metabolic experiments.
• Gene mutations help explain metabolic disorders and enzyme regulation.
Each level provides different insight — from whole-body physiology to molecular mechanisms.
Allows metabolic study of a single organ without systemic interference.
They maintain organelle integrity and preserve native tissue architecture.
To identify the path and rate at which substrates flow through metabolic pathways.
Immortal cells enable consistent and reproducible metabolic studies.
They reveal gene defects responsible for metabolic disorders and pathway regulation.
Tissue culture is the maintenance and growth of isolated cells, tissues, or organs outside the organism in a controlled, nutrient-rich environment.
• Appropriate culture medium (amino acids, glucose, vitamins, salts)
• Physiological pH (~7.2)
• Temperature control (usually 37°C)
• Sterile conditions
• CO₂ incubator to maintain buffering
• Aseptic handling environment (laminar airflow)
Cells obtained directly from fresh tissues.
• Limited lifespan
• Most closely resemble in-vivo cells
Cells that can proliferate indefinitely.
Examples: HeLa cells, hybridoma cells
• Used extensively in biochemical and drug studies
Small tissue fragments cultured while preserving architecture.
• Useful for studying organ-specific metabolism and histological effects.
Cells metabolize labelled substrates
• e.g., radiolabelled glucose → CO₂, glycogen, lipids
Useful to map pathways like glycolysis, TCA, biosynthesis.
Cells incorporate radiolabelled precursors
• e.g., labelled thymidine → DNA
• labelled uridine → RNA
Evaluate:
• Drug metabolism
• Cytotoxicity
• Growth inhibitory effects
• Anticancer screening
• Monoclonal antibodies (using hybridoma technology)
• Therapeutic proteins
• Vaccine production
Viruses must grow inside living cells → tissue culture is essential for:
• Viral multiplication
• Testing antiviral drugs
• Vaccine development
• Gene transfection
• Gene expression studies
• Production of recombinant proteins
• CRISPR editing studies
Cancer cell lines help study:
• Uncontrolled cell division
• Metastasis
• Gene mutations
• Chemotherapeutic responses
• Highly controlled environment
• Reproducible results
• Easy manipulation of nutrients, hormones, drugs
• Direct observation of cell behavior
• Enables growth of viruses (cannot be grown in cell-free systems)
• High skill and aseptic technique required
• Expensive equipment
• Contamination risk
• Cells may behave differently from whole tissue environment
• Tissue culture = growth of cells in artificial medium.
• HeLa cell line = classic immortal cancer cell line.
• Used for metabolism studies, drug testing, DNA synthesis, virology.
• Hybridoma cells → monoclonal antibodies.
• Culture medium must maintain pH ~7.2 and provide amino acids, glucose, vitamins.
• Used in genetic engineering and recombinant protein production.
• Offers excellent control but limited ability to mimic whole-organ physiology.
Because it allows direct measurement of substrate uptake, metabolic flux, and enzyme activity in living cells.
Primary cells have limited lifespan; continuous cell lines can proliferate indefinitely.
It is the first immortal human cell line, widely used for metabolic and drug studies.
Most cells require pH ~7.2; even small deviations affect enzyme function and survival.
Fusion of B-cells with myeloma cells to produce monoclonal antibodies.
Viruses require living cells to replicate, so tissue culture is essential for their study.
Radioisotope tracers are radiolabelled atoms or molecules used to follow the path, rate, and fate of metabolic processes inside living cells, organs, or organisms.
They allow direct tracking of precursor → product relationships.
• Incorporate naturally into metabolic pathways
• Emit detectable radiation → easy to track
• Reveal metabolic pathways, turnover, and organ distribution
• Help measure synthesis and degradation rates
• Identify metabolic blocks in disease
Radioisotopes act as “metabolic markers.”
Used for:
• Carbohydrate metabolism
• Lipid synthesis
• Amino acid incorporation
• Following fate of labelled glucose
Used for:
• Protein turnover
• Nucleotide and nucleic acid synthesis
• Heme synthesis
Used for:
• ATP production
• Phosphorylation studies
• DNA/RNA synthesis
• Kinase activity
Used for:
• Thyroid metabolism and hormone uptake
Used for:
• DNA replication
• Lipid membranes
• Hormone binding studies
Example:
• ¹⁴C–glucose → measure labelled CO₂ → map glycolysis & TCA
• ¹⁴C–acetate → trace lipid biosynthesis
Used to calculate rates of synthesis and breakdown:
• Proteins
• Nucleic acids
• Glycogen
• Lipids
Feeding, infusion, or injection of labelled compounds:
• Track where they accumulate
• Identify which organ metabolizes them fastest
(example: liver → highest glucose metabolism)
Inborn errors of metabolism show:
• Failure to convert labelled substrate to expected product
• Accumulation of certain intermediates
Labelling cells with:
• ³H-thymidine → DNA replication
• ³H-uridine → RNA synthesis
• ¹⁴C-amino acids → protein synthesis
Used to study cell cycle and drug effects.
⁵⁹Fe-labeled iron allows measurement of:
• RBC lifespan
• Heme turnover
• Iron storage disease evaluation
• Highly sensitive
• Very small quantities needed
• Allows real-time metabolic tracking
• Can be used in vivo and in vitro
• Quantitative measurements possible
• Radioactive waste handling
• Potential radiation hazard
• Expensive detectors
• Requires specialized laboratory
• Radiolabels help trace metabolic pathways and reaction sequences.
• ¹⁴C → carbohydrate + lipid metabolism.
• ¹⁵N → protein and nucleic acid studies.
• ³²P → ATP turnover, kinases, DNA/RNA synthesis.
• ³H-thymidine → DNA replication marker.
• ⁵⁹Fe → RBC lifespan and heme metabolism.
• Radioisotopes help identify metabolic blocks in genetic diseases.
• Critical for research in cell cycle, oncology, endocrinology, hematology.
Because they allow direct tracking of molecules through metabolic pathways.
It helps trace carbohydrate metabolism all the way to CO₂.
It labels ATP and phosphorylated intermediates → used to study energy metabolism.
As a marker for DNA synthesis and cell proliferation.
A defective pathway shows failure of conversion of labelled precursor to expected product.
To study RBC lifespan and iron turnover.
Some can (like iodide uptake in thyroid), but most are done in cells, animals, and isolated tissues due to safety concerns.
Different organs have distinct metabolic preferences depending on their function and physiological state. Fuel choice changes in fed, fasting, and starvation periods to maintain energy homeostasis.
• Requires 10–20% of cardiac output despite being only 2% of body weight.
• Depends almost entirely on glucose in the fed state.
• Cannot utilize fatty acids because they do not cross the blood–brain barrier.
• During prolonged starvation, uses ketone bodies, reducing glucose requirement.
• Extremely sensitive to hypoglycemia.
• Central organ for carbohydrate, fat, and protein metabolism.
• Stores glycogen post-meal.
• Performs fatty acid synthesis and secretes VLDL.
• Major site of amino acid degradation and urea cycle.
• Fasting state: performs glycogenolysis followed by gluconeogenesis.
• Produces ketone bodies for peripheral tissues but does not use ketones itself.
• At rest → uses fatty acids predominantly.
• During intense activity → uses muscle glycogen, later shifts back to fatty acids as exercise continues.
• During fasting → uses fatty acids and ketone bodies.
• During prolonged starvation → metabolizes branched-chain amino acids.
• Contains the phosphocreatine system for rapid ATP generation.
• Extremely energy-dependent; consumes large amounts of ATP daily.
• Primarily uses fatty acids (60–90%) for energy.
• Also utilizes glucose and ketone bodies.
• Relies on the creatine kinase shuttle to deliver ATP to myofibrils.
• In heart failure, shifts toward higher glucose use.
• Stores triglycerides, which form nearly 85% of the body’s total fuel reserve.
• Releases free fatty acids during fasting through hormone-regulated lipolysis.
• Insulin inhibits lipolysis; glucagon and catecholamines stimulate it.
• Continuous turnover occurs even in fed state.
• Performs significant gluconeogenesis during prolonged fasting.
• Utilizes fatty acids and glutamine for energy.
• Produces ammonia to maintain acid–base balance.
• Absorbs dietary glucose, amino acids, and fatty acids.
• Converts a portion of glucose to lactate, which enters the liver for the Cori cycle.
• Enterocytes use glutamine as their primary fuel.
• Lack mitochondria → rely exclusively on anaerobic glycolysis for ATP.
• Produce lactate as end product.
• Require NADPH from the HMP shunt to protect against oxidative stress.
Skeletal muscle: shifts to fatty acids, ketone bodies, and branched-chain amino acids.
Heart: continues using fatty acids and ketone bodies.
Brain: begins using ketone bodies after ~3 days.
Liver: increases gluconeogenesis and ketogenesis.
Adipose tissue: increases lipolysis.
• Each organ has a unique metabolic preference based on physiology.
• Brain uses glucose; shifts to ketones only during prolonged fasting.
• Heart relies heavily on fatty acids.
• Skeletal muscle uses glycogen during exercise; fatty acids during rest and fasting.
• Liver is the main site for gluconeogenesis, ketogenesis, and urea synthesis.
• RBCs depend only on glycolysis.
• Kidney contributes to gluconeogenesis in starvation.
• Adipose tissue is the major storage site of body fuel.
Fatty acids cannot cross the blood–brain barrier.
Liver lacks the enzyme thiophorase, required to utilize ketones.
Fatty acids provide the highest ATP yield and support continuous contractile activity.
They lack mitochondria, so oxidative pathways are not possible.
Fatty acids, ketone bodies, and branched-chain amino acids.
Glutamine, not glucose.
• Each organ has a characteristic fuel preference based on its function.
• Brain relies almost entirely on glucose; shifts to ketone bodies only after prolonged fasting.
• Heart uses fatty acids as its major energy source; ketone use increases in fasting.
• Skeletal muscle uses glycogen during exercise; fatty acids during rest and fasting; BCAA oxidation increases in starvation.
• Liver regulates blood glucose via glycogen storage, glycogenolysis, and gluconeogenesis.
• Liver produces ketone bodies but can’t use them because it lacks thiophorase.
• RBCs use only anaerobic glycolysis (no mitochondria) and rely on NADPH to prevent oxidative stress.
• Kidney contributes significantly to gluconeogenesis in prolonged fasting and helps regulate acid–base balance via ammonia production.
• Adipose tissue stores triglycerides and releases fatty acids during fasting through hormone-sensitive lipolysis.
• Intestine uses glutamine as its primary fuel and releases lactate into portal circulation.
• During starvation, the body shifts from glucose to fatty acids and ketone bodies to spare muscle protein.
• Brain ketone use reduces glucose demand and slows muscle proteolysis.
• Heart failure alters fuel preference—shifts from fatty acids to more glucose utilization.
Since the brain depends heavily on glucose, prolonged hypoglycemia can cause confusion, seizures, coma, and irreversible neuronal injury.
Conditions: insulin overdose, alcoholism, liver failure.
Excess fatty acid oxidation → massive ketone production.
Brain adapts but acidosis produces dehydration, altered sensorium, and rapid respiration.
Occurs in Type 1 diabetes due to insulin deficiency.
When oxygen is low, the heart cannot use fatty acids and switches abruptly to anaerobic glycolysis → lactate accumulation → pain and reduced contractility.
Seen in angina, heart attacks.
Intense exercise uses glycogen; depletion leads to fatigue.
Lactic acid accumulation temporarily lowers pH → muscle ache.
In prolonged endurance exercise, muscle shifts to fatty acid oxidation.
Skeletal muscle cannot break down glycogen due to myophosphorylase deficiency.
Causes exercise intolerance, muscle cramps, and “second wind” phenomenon.
Damaged liver cannot perform gluconeogenesis → hypoglycemia.
Impaired urea cycle → ammonia accumulation → hepatic encephalopathy.
During prolonged starvation, the body increases ketone production.
If starvation persists, skeletal muscle proteolysis increases to supply amino acids for gluconeogenesis.
Alcohol metabolism increases NADH → inhibits gluconeogenesis.
Leads to hypoglycemia, fatty liver, lactic acidosis.
Kidney metabolism fails to generate enough ammonia for acid–base balance → metabolic acidosis.
Seen in Type 2 (proximal) and Type 1 (distal) RTA.
G6PD deficiency → reduced NADPH → hemolysis after oxidative stress (drugs, fava beans, infections).
Symptoms: jaundice, anemia, dark urine.
In malnutrition or prolonged fasting, reduced glutamine supply weakens enterocytes → impaired absorption, diarrhea, increased infection risk.
Heart reduces fatty acid oxidation and uses more glucose due to insulin resistance.
Patients show fatigue, reduced exercise capacity.
A. Fatty acids
B. Glucose
C. Ketone bodies
D. Amino acids
A. Lactate
B. Glutamine
C. Ketone bodies
D. Propionate
A. HMG-CoA synthase
B. Thiophorase
C. Carnitine acyltransferase
D. Pyruvate carboxylase
A. Glucose
B. Ketone bodies
C. Fatty acids
D. Pyruvate
A. Brain
B. Intestinal mucosa
C. RBC
D. Heart
A. Fatty acids
B. Glycogen
C. Ketone bodies
D. Amino acids
A. Glucose
B. Glutamine
C. Ketone bodies
D. Palmitate
A. Lactate
B. Amino acids
C. Free fatty acids
D. Glycerol only
A. Heart
B. Intestine
C. Kidney
D. Brain
A. Transport glucose
B. Facilitate ATP delivery to myofibrils
C. Increase lactate production
D. Store glycogen
A. Liver
B. Brain
C. Intestine
D. Heart
A. Glucose
B. Ketone bodies
C. Fatty acids
D. Glycogen
A. Ketone bodies
B. Glycerol
C. Branched-chain amino acids
D. Cholesterol
A. Liver glycogen
B. Skeletal muscle glycogen
C. Plasma glucose
D. Adipose triglycerides
A. Kidney
B. Intestine
C. Adipose tissue
D. Brain
1-B
2-C
3-B
4-C
5-C
6-B
7-B
8-C
9-C
10-B
11-C
12-C
13-C
14-D
15-B
Glucose.
Fatty acids cannot cross the blood–brain barrier.
Ketone bodies.
It reduces glucose demand and spares muscle protein.
Fatty acids.
Yes — especially during fasting and prolonged exercise.
Fatty acid oxidation yields maximum ATP, needed for continuous contraction.
Thiophorase (succinyl-CoA:acetoacetate CoA transferase).
Glycogenolysis → gluconeogenesis → ketogenesis.
Fatty acids.
Muscle glycogen (via anaerobic glycolysis).
Branched-chain amino acids (leucine, isoleucine, valine).
Phosphocreatine system.
Glutamine.
Intestine.
Anaerobic glycolysis.
They lack mitochondria.
Hexose monophosphate (HMP) shunt.
Adipose triglycerides.
Free fatty acids (and glycerol).
Kidney.
Glutamine.
Helps maintain acid–base balance.
Through the creatine kinase shuttle.
Around 3 days of starvation.
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