Lecture: Fluxes and Flows in Metabolism

# Fluxes and Flows in Metabolism ## Ask me Anything - 0 questions - 0 upvotes ## Fluxes and flows in metabolism - Bas Teusink ## What metabolic state is this? This is a bar graph comparing the metabolic state of a person in 3 different conditions: - **Overnight fast:** The state of a person...

# Fluxes and Flows in Metabolism ## Ask me Anything - 0 questions - 0 upvotes ## Fluxes and flows in metabolism - Bas Teusink ## What metabolic state is this? This is a bar graph comparing the metabolic state of a person in 3 different conditions: - **Overnight fast:** The state of a person after a long night without food. - **Post-prandial:** The state of a person after a meal. (This is usually called "fed state"). - **Starved:** The state of a person after prolonged deprivation of food. The height of each bar represents the number of people in each condition. - **Overnight fast:** 8 people - **Post-prandial:** 19 people - **Starved:** 0 people The bar graph indicates that most of the people are in a post-prandial state. The image contains a schematic diagram of a human metabolic state, with the following components: - **Blood:** - Insulin - Glucagon - **Liver:** - Glucose - Glycogen - Acetyl CoA - TCA (Tricarboxylic acid cycle) - ATP - CO2 - TG (Triglycerides) - **Adipose:** - Glucose - TG (Triglycerides) - Glycerol - **VLDL:** (Very low-density lipoprotein) - FA (Fatty acid) - Glycerol The numbers in the image indicate different metabolic processes occuring in the body. ## Postprandrial state (fed state) - **Nutrients are stored:** - Glycogen - Protein mass - Fat: triglycerides - **Insulin high, glucagon low** This is another schematic diagram of the human body, this time depicting the **postprandrial** metabolic state. The image contains the following components: - **Intestine:** - CHO (Carbohydrates) - Fat (TG) - Protein - Chylomicrons (TG) - AA (Amino acids) - **Blood:** - Insulin - Glucose - Glucagon - **Liver:** - Glucose - Glycogen - Acetyl CoA - TCA - ATP - CO2 - TG - **Adipose:** - Glucose - TG - Glycerol - **VLDL:** - FA + Glycerol - **RBC:** (Red blood cells) - **Pyruvate** - **Lactate** - **Brain:** - Acetyl CoA - TCA - CO2 - ATP - **Muscle:** - Acetyl CoA - TCA - CO2 - ATP - Glycogen - Glucose The numbers in the image indicate different metabolic processes occuring in the body. ## Basal state (overnight fast) - **Stores mobilized:** - Glycogen for glucose - Protein for glucose - Fat for energy - **Insulin low, glucagon rising** This is a third schematic diagram, this time depicting the **basal** metabolic state. The image contains the following components: - **Blood:** - Glucose - Insulin - Glucagon - **Liver:** - Glycogen - Glucose - ATP - Acetyl CoA - KB (Ketone bodies) - **Adipose:** - TG - FA - KB - **VLDL:** - KB - AA - FA - Acetyl CoA - TCA - CO2 - ATP - **Muscle:** - AA - Protein - Acetyl CoA - TCA - CO2 - ATP - **RBC:** - Lactate - **Brain:** - Acetyl CoA - TCA - CO2 - ATP - Glucose - **Kidney:** - Urea - Urine The numbers in the image indicate different metabolic processes occuring in the body. Key information is highlighted in **Red**: "Red: not in the original Marks' figure." ## Towards metabolic pathways and flow This is a metabolic pathway map depicting crucial metabolic pathways that occur within living organisms. The image contains the following components: - **Glycogen Metabolism** - **Pentose Phosphate Pathway** - **Non-growth Associated Energy Maintainance** - **Malonyl CoA Synthesis** - **Lactate** - **Glycerol Phosphate Shuttle** - **TCA Cycle** - **Electron Transport Chain** - **Malate Aspartate Shuttle** The metabolic map shows interconnected pathways, involving various metabolic substrates and enzymes. The **TCA cycle** is highlighted in a large circle. ## Equilibrium versus steady state - **Example of anti cancer drug** - **No flow: ∆G=0, Equilibrium** - **Flow: ∆G<0 (2x), Steady state ** This diagram illustrates the concept of **equilibrium** and **steady state**. The image depicts 2 scenarios of a **cancer cell** in a **blood** environment: - **Scenario 1- Equilibrium** - A **cancer cell** is submerged in a **blood** environment, with no flow of **anti cancer drug** between the two. This condition is characterized by a **∆G=0**, indicating that the system is at **equilibrium**, with no flow of the **anti cancer drug** between the cell and the environment. - **Scenario 2 - Steady state** - A **cancer cell** is submerged in a **blood** environment with a **multidrug resistance pump** present in the cell. This pump continuously expels **anti cancer drug** back into the blood despite the drug diffusing from the blood into the cell. This condition is characterized by a continuous flow of the **drug** between the cell and the environment, and a **∆G<0**, indicating that the cell is in a **steady state**. ## Steady state versus equilibrium - **Make a balance for a variable of the system** - **System is cell** - **Variable is concentration of Drug (D)** - **∆D = ∆¡D + ∆D** This diagram elaborates further on the concept of **steady state** in a broader context, beyond the example of **anti cancer drug**. The image emphasizes: - The **concentration of drug (D)** is a variable of the system, and can change. - The change in **drug concentration** within the system is represented by **∆D**: - **∆¡D** represents **exchange** of the drug between the system and the environment - **∆D** represents the **production** or **consumption** inside the system. The image continues to illustrate how the concept of **steady state** can be applied in a broader context beyond the example of **anti-cancer drug**. ## With pump: steady state - **Make a balance for a variable of the system** - **System is cell** - **Variable is concentration of Drug (D)** - **There are two independent exchange processes. Independent means that each has its own ∆G.** - **∆D = ∆¡D + ∆D** - **= ∆ (MDR pump) + ∆』(diffusion) + 0** - **No internal breakdown of the drug** - **Now suppose ∆』(MDR pump) = - 150 nmol/h and ∆』(diffusion) = + 150 nmol/h: - **∆D = 0. The drug concentration in the system is balanced: it is in steady state**.** This diagram explores the concept of **steady state** further, focusing on the dynamics of **drug concentration** in a **cell** with a **multidrug resistance pump**. - The **pump** and **diffusion** are independent processes, with their own **∆G**. - The diagram provides a more detailed mathematical approach to illustrate how the **concentration of drug** in the system is balanced: - The **pump** transports **drug** out of the cell with a rate of **- 150 nmol/h**, while - The **diffusion** process transports **drug** into the cell with a rate of **+ 150 nmol/h**. - In this scenario, the change in drug concentration **∆D** is zero, indicating a **steady state** where the **drug concentration** remains constant. ## Steady-state fluxes between organs - **Erythrocyte** - **Brain** - ATP - Glucose - **Liver** - Ketone bodies - **Adipocyte** - Fatty acids - ATP - **Skeletal muscle** - **Glucose in system blood** - **∆glucose = ∆glucose + ∆glucose ** - **= ∆ liver (>0) + ∆ brain (<0) + ∆₁ erythrocyte (<0)** This diagram illustrates the concept of **steady state fluxes** between different organs of the body. - **Steady state fluxes** refers to the constant exchange of molecules between the body's different organs. - The diagram depicts a scenario where **glucose** is being exchanged between: - **Brain**, which utilizes **glucose** for energy production. - **Liver**, which can either store **glucose** as **glycogen** or produce **glucose** through **gluconeogenesis** - **Skeletal muscle**, which can either store **glucose** as **glycogen**, or utilize it for energy production. - **Adipose tissue**, which can either store **glucose** as **triglycerides**, or release **fatty acids** for energy production. - **Erythrocytes**, which use **glucose** as a source of energy. - The net flux of **glucose** is determined by adding the contributions from each individual organ, resulting in a **positive** flux from **liver** to **brain**, **adipose**, and **skeletal muscle**, and a **negative** flux from **erythrocytes** to **brain** and **adipose**. ## Steady-state fluxes in metabolic pathways - **Portion isomerized from aldehyde to keto sugar** - **AGlucose 6-phosphate / At** - **= rate of Hexokinase - rate of Phosphoglucose isomerase** - **= 0** - **AFructose 6-phosphate / At ** - **= rate of Phosphoglucose isomerase – rate of Phosphofructokinase** - **= 0** - **Etc...** - **Concentrations are constant in time, but there is continuous flow. We call this steady-state flux** This schematic illustration depicts the **steady-state flux** in the **glycolysis** metabolic pathway. - The diagram illustrates the individual **reaction steps** in **glycolysis**, from the conversion of **Glucose** to **Pyruvate**. - The diagram shows the following enzymatic reactions: - **Hexokinase** - **Phosphoglucose isomerase** - **Phosphofructokinase** - **Aldolase** - **Triose phosphate isomerase** - **Glyceraldehyde 3-phosphate dehydrogenase** - **Phosphoglycerate kinase** - **Phosphoglycerate mutase** - **Enolase** - **Pyruvate kinase** - The diagram emphasizes that: - While the **concentrations** of intermediate metabolites remain **constant** throughout the **glycolysis** pathway in a **steady state**, - There is a **continuous flow** of molecules through each step of the pathway. ## How is bodyweight (BW) regulated? - **Energy expenditure** - **Energy e** - **En** This diagram presents a bar graph to demonstrate the concept of **bodyweight regulation**: - The bar graph represents 3 individuals: **A, B, and C**. - The height of each bar represents the **bodyweight** of each individual. - **Individual A** has the lowest bodyweight of **13**. - **Individual B** has the highest bodyweight of **22**. - **Individual C** has a bodyweight of **1**. The image includes a graph depicting the relationship between **energy expenditure, energy intake**, and **bodyweight**. - The graph illustrates that: - **Energy expenditure** decreases with **increasing bodyweight**. - **Energy intake** increases with **increasing bodyweight**. - The **intersection** of the **energy expenditure** and **energy intake** lines determines the **equilibrium bodyweight** of the individual. ## The Lancet 2011 (!) - **Obesity 3** - **Quantification of the effect of energy imbalance on bodyweight** - **Kevin D Hall, Gary Sacks, Dhruva Chandramohan, Carson C Chow, Y Claire Wang, Steven L Gortmaker, Boyd A Swinburn** - **Key messages:** - **Health and nutrition organizations have perpetuated the myth that a reduction of food intake of 2 MJ per day will lead to a steady rate of weight loss of 0.5 kg per week.** - **Because this static weight-loss rule does not account for dynamic physiological adaptations that occur with decreased bodyweight, its widespread use at both the individual and population levels has led to drastically overestimated expectations for weight loss.** This introductory slide references a publication in "The Lancet", focusing on the topic of **obesity**. It highlights the following key points: - It criticizes the common misconception that **reducing caloric intake by 2 MJ (megajoules) per day** will consistently result in **0.5 kg of weight loss per week**. - The slide emphasizes that this **static weight-loss rule** overlooks the **dynamic physiological adaptations** that the body undergoes in response to **weight loss**. - Therefore, it emphasizes that the general public has **overestimated expectations** about **weight loss** through calorie restriction alone. The slide also includes a graph depicting the relationship between **energy expenditure, energy intake**, and **bodyweight**: - This graph illustrates that: - **Energy expenditure** is **higher** at lower **bodyweight**. - **Energy intake** is **lower** at lower **bodyweight**. The slide concludes by arguing that **effective weight management** requires a more nuanced approach that acknowledges the **dynamic physiological adaptations** that occur with weight loss. ## The false static picture - **Energy expenditure** - **Decrease in energy intake leads to continuous decrease in BW** - **Energy intake** This slide contrasts the **false static picture** of weight loss with the reality of **dynamic physiological adaptations** that occur with weight loss. It highlights the following points: - The **false static picture** assumes that reducing **energy intake** will lead to a **continuous decrease in bodyweight**. - This is akin to assuming that **energy expenditure** remains constant, regardless of **bodyweight**. - In reality, the body undergoes **dynamic physiological adaptations** in response to **reduced energy intake**. - These adaptations include a **decrease in energy expenditure**, which can **slow down** weight loss. ## The Lancet 2011 (!) - **Obesity 3** - **Quantification of the effect of energy imbalance on bodyweight** - **Kevin D Hall, Gary Sacks, Dhruva Chandramohan, Carson C Chow, Y Claire Wang, Steven L Gortmaker, Boyd A Swinburn** - **Key messages:** - **Health and nutrition organizations have perpetuated the myth that a reduction of food intake of 2 MJ per day will lead to a steady rate of weight loss of 0.5 kg per week.** - **Because this static weight-loss rule does not account for dynamic physiological adaptations that occur with decreased bodyweight, its widespread use at both the individual and population levels has led to drastically overestimated expectations for weight loss.** - **On the basis of our model, we propose an approximate rule of thumb for an average overweight adult: every change of energy intake of 100 kJ per day will lead to an eventual bodyweight change of about 1 kg (equivalently, 10 kcal per day per pound of weight change) with half of the weight change being achieved in about 1 year and 95% of the weight change in about 3 years.** This slide references a publication in "**The Lancet, again. It expands on the key messages, highlighting the following points:**** - The slide again emphasizes the **myth** that reducing **food intake by 2 MJ per day** will lead to **0.5 kg of weight loss per week**. - The slide clarifies that the **proposed static weight-loss rule** lacks the necessary considerations for the **dynamic physiological adaptations** that occur with **decreased bodyweight**. - Finally, the slide suggests a more accurate **rule of thumb** for an **average overweight adult:** that every **100 kJ (kilojoules) per day change in energy intake** will result in an **approximate 1 kg change in bodyweight** over a longer timeframe. The slide also includes a graph depicting the relationship between **energy expenditure, energy intake**, and **bodyweight**: - This graph illustrates that: - **Energy expenditure** is **higher** at lower **bodyweight**. - **Energy intake** is **lower** at lower **bodyweight**. The slide concludes by emphasizing the importance of understanding the **dynamic physiological adaptations** that occur with weight loss, and proposing a more accurate rule-of-thumb for weight loss management. ## Need for fast(er) flux regulation - **Heat** - **CO2** - **ATP** - **Energy production via oxidation of:** - **Carbohydrate** - **Lipid** - **Protein** - **O2** - **Energy utilization:** - **Biosynthesis** - **Detoxification** - **Muscle contraction** - **Active ion transport** - **Thermogenesis** - **ADP + Pi** This slide elucidates the essential role of **flux regulation** in maintaining metabolic processes. This image illustrates the complex interplay between energy production and utilization in living organisms. - It highlights the role of **ATP**(as the energy currency of the cell) and **ADP** (adenosine diphosphate) in cellular energy transactions. - The diagram also emphasizes the critical interplay between **energy-producing pathways**, including **oxidation** of **carbohydrates, lipids, and proteins**, and **energy-consuming pathways**, involving various cellular functions such as **biosynthesis, detoxification, muscle contraction, active ion transport**, and **thermogenesis**. ## Which parameter changes through regulation by gene expression? - **[E]** - **k_cat** - **Km** - **[S]** This slide presents a bar graph that explores the different parameters that are affected by **gene expression** regulation. - The bar graph represents four different parameters that influence enzyme activity: - **[E]** = **Enzyme concentration** - **k_cat** = **Turnover number** - **Km** = **Michaelis constant** - **[S]** = **Substrate concentration** The height of each bar represents the number of people who voted for that parameter being affected by **gene expression**. - **[E]** receives the highest number of votes (14), indicating that people believe **gene expression** primarily influences the **concentration of enzyme**. - The other three parameters receive significantly fewer votes, suggesting they are less affected by **gene expression** regulation. ## How to regulate a metabolic rate? - **v = kcat [E] [S] / [S] + KM** - **Gene expresion** - **Post-translational modifications** - **Metabolic regulation, allosteric regulation** This summary slide reviews the different ways in which metabolic rates can be regulated, encompassing three main mechanisms: - The slide introduces the **Michaelis-Menten equation** as the foundation for understanding enzyme kinetics. - The equation describes the relationship between the **reaction rate (v)**, the **enzyme concentration ([E])**, the **substrate concentration ([S])**, the **turnover number (kcat)** and the **Michaelis constant (Km)** - The slide then highlights **three key mechanisms** for regulating enzyme activity and affecting the **reaction rate**: - **Gene expression** influences the **enzyme concentration ([E])**, by controlling the production of new enzymes. - **Post-translational modifications** can alter the catalytic activity of enzymes by modifying their structure or function. - **Metabolic regulation, including allosteric regulation**, involves the interactions of molecules with enzymes, either activating or inhibiting them. ## At what time scale does this process take place? - **post-translational modification** - **22.7 seconds** - **allosteric regulation** - **14.7 seconds** - **gene expression regulation** - **38.7 hours** The slide emphasizes the temporal differences between different regulatory mechanisms, using a bar graph to compare the time scales involved: - **Post-translational modifications** occur within the **shortest timeframe** (seconds), meaning they can provide rapid responses in metabolic regulation. - **Allosteric regulation** follows closely behind, also occurring in seconds, enabling swift adaptive adjustments. - **Gene expression regulation**, however, takes a much longer time (hours), suggesting it plays a role in long-term metabolic changes rather than immediate responses. ## PTM and metabolic regulation (MR) both act at seconds. What is the difference? - **MR regulates internal communication, PTM external** - **MR acts on catabolism, PTM on anabolism** - **MR uses ATP, PTM uses phosphate** This bar graph compares **post-translational modifications (PTM)** and **metabolic regulation (MR)**, both fast-acting mechanisms. - The slide outlines the key differences: - **MR** primarily regulates internal communication, often involving pathways that break down molecules. - **PTM** primarily regulates external communication, often involving biosynthetic pathways that build up molecules. - **MR** typically uses **ATP** as an energy source, while **PTM** typically uses **phosphate** for modification. ## What is true? - **hexokinase is sensitive to [glucose]** - **hexokinase is insensitive to [glucose]** The slide presents two competing statements about the enzyme **hexokinase** in a bar graph format. - The bar graph provides votes to indicate the degree of consensus for each statement. - **Hexokinase is insensitive to glucose concentration** receives a higher number of votes (23), suggesting it is a widely accepted notion. ## Which enzymes to regulate? This image is a complex metabolic map, depicting the intricate network of interconnected pathways within living organisms. - The map includes various metabolic reactions, enzymes, cofactors, and the corresponding pathways they contribute to. - The central focus of the map is to identify **key enzymes** that act as **rate-limiting steps** or **regulators** of critical metabolic pathways. ## Which enzyme would be a good target of regulation? - **PGM** - **TIM** - **Aldolase** - **PFK** This image is a bar graph representing the **free energy change (∆G⁰')** for different enzymatic reactions in **Glycolysis**. - The bar graph depicts the **∆G⁰'** values for **four key enzymes** in **glycolysis**: - **PGM**: Phosphoglycerate Mutase - **TIM**: Triose Phosphate Isomerase - **Aldolase**: Fructose-1,6-bisphosphate Aldolase - **PFK**: Phosphofructokinase-1 - **PFK** has the most **negative ∆G⁰'** (-21), suggesting that it is highly thermodynamically favorable to proceed in the forward direction and serves as an important regulatory point in the pathway. ## Which column determines which enzymes are "pumps"? - **DeltaG⁰'** - **DeltaG** - **either** This image is a continuation of the previous slide, comparing the **free energy change (∆G⁰')**, a standard measure of reaction feasibility, and the **actual free energy change (∆G)**, which considers the actual conditions. - **DeltaG** is the key criterion for identifying which enzymes are the "pumps". - **"Pumps"** are enzymes that drive a reaction forward by having a negative **DeltaG**, often due to the coupling with ATP hydrolysis. ## Irreversible enzymes are used as "pumps" - **Table 14-1 ∆G°' and ∆G for the Reactions of Glycolysis in Heart Muscle** - **Reaction** - **Enzyme** - **AG°' (kJ·mol−¹) ** - **AG (kJ·mol−¹) ** This slide showcases a table summarizing the **thermodynamic parameters (∆G°' and ∆G)** for the enzymes involved in **Glycolysis**. - The table illustrates the concept that enzymes with a significant, negative **∆G°'** are considered irreversible. - **Irreversible enzymes** serve as important regulatory points in metabolic pathways: - Their function is crucial for pushing a metabolic pathway in a specific direction and driving a step forward. ## Glycolysis makes ATP: ATP inhibits pump PFK - **Glucose** - **ATP** - **Hexokinase** - **Glucose 6-P** - **Fructose 6-P** - **ATP** - **Phosphofructokinase-1** - **Fructose-1,6-bis P→ 2 NADH → 4 ATP** - **Pyruvate** - **Shuttle system** - **Electron transport chain** - **H+** - **O2** - **CO2** - **H2O** - **ADP + Pi** - **ATP** - **Mitochondrion** - **ATP synthase** - **H+** - **Fig 22.1** - **Table 14-1 ∆G°' and ∆G for the Reactions of Glycolysis in Heart Muscle** - **Reaction** - **Enzyme** - **AGO (kJ·mol ¹)** - **AG (kJ·mol ¹)** - **B** - **+ AMP or fructose 2,6-bis-P** - **V** - **Vmax** - **ATP (mM)** - **Fig 22.16B** This image continues to explore how **ATP** can regulate the speed of **Glycolysis**. - This image illustrates that: - A key regulatory enzyme in **Glycolysis** is **Phosphofructokinase 1 (PFK1)**, which catalyzes the committed step in **Glycolysis** from **Fructose 6-Phosphate** to **Fructose 1,6-bisphosphate**. - **PFK1** plays a crucial role in **regulating Glycolysis**, as it is a highly regulated and irreversible enzyme. - The activity of **PFK1** is sensitive to the concentration of **ATP**, which is a product of **Glycolysis**. - **ATP** acts as an **inhibitor** of **PFK1**, ensuring that when **ATP** levels are already high, **Glycolysis** is not activated further. ## How to regulate metabolic rate? - **1. Regulation of enzymes at 3 levels** - **Metabolic (allosteric) interactions (sec-min; intracellular)** - **Post-translational via signaling of nutrients, hormones or neurons (sec-min; inter-organ)** - **Enzyme levels via gene expression (h-days; intracellular)** - **2. Organ-specific expression of iso-enzymes and transporters** - **3. View pathways from the perspective of their function (which can be organ-specific): often negative feedback is used** This slide summarizes the different mechanisms of metabolic rate regulation at a higher level. It outlines three key strategies: - Regulation of enzymes at 3 levels: - **Metabolic (allosteric) interactions**, operating within seconds to minutes within cells, allowing for rapid response to the environment. - **Post-translational modifications**, also operating within seconds to minutes, can be triggered by nutrient availability, hormone signaling, or neuronal activity. - **Enzyme levels via gene expression**, taking hours to days, influencing long-term metabolic changes. - **Organ-specific expression of iso-enzymes and transporters**, allowing for specialized metabolic functions in different organs. - By viewing pathways from the perspective of their function, a deeper understanding is gained. This approach often utilizes **negative feedback mechanisms**, where a product of a pathway inhibits the pathway's activity to maintain a stable balance. ## What is the function of respiration? - **NADH production** - **ATP production** - **reactive oxygen species removal** This bar graph serves as a quiz to assess understanding of the core function of **respiration**. - **Respiration** is the process of converting energy from nutrients into a form usable by cells, primarily **ATP**. - The bar graph reflects that: - **ATP production** is the primary function of respiration. - While **NADH production** and **reactive oxygen species removal** are associated with respiration, they are not its fundamental goal. ## How is respiration regulated? - **Option1** - **Option 2** - **Option 3** This bar graph examines the mechanisms by which **respiration** is regulated. - It presents three options and asks which one is the most accurate representation. - **Option 2** receives the most votes, highlighting the relationship between **ATP concentration** and **respiration rate**. ## What principle is the basis of homeostasis? - **allosteric regulation** - **negative feedback** - **positive feedback** This slide focuses on the crucial principle underlying **homeostasis**, the ability of a system to maintain a stable internal environment. - The bar graph indicates that **negative feedback** is the primary principle governing homeostasis for the majority of respondents (1). It is considered a fundamental mechanism, as it involves a product of a pathway inhibiting the pathway's activity, preventing excessive production and maintaining a balance within the system. ## Conclusions - **Regulation of fluxes is key to adaptation (and disease)** - **Regulation can only be understood from the perspective of function** - **Products of a pathway often inhibit the pumps of the pathway** - **Different ways of regulation: time-scales and intra or inter cellular coordination** This final slide summarizes the key takeaways from the presentation: - **Flux regulation**, controlling the rate of flow through metabolic pathways, is essential for biological adaptation and plays a significant role in preventing disease. - It can't be understood by isolating individual pathways; instead, it requires a systematic view of a pathway's purpose and function within the larger metabolic network. - **Negative feedback mechanisms** are prevalent in metabolic regulation, where the products of a pathway often inhibit the enzymes responsible for the production of those products. - Finally, the slide highlights the different **time scales** and **cellular coordination** involved in regulating metabolic fluxes, emphasizing the complexity and interconnectedness of metabolism. 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