Chapter 2

Principles of Intermediary Metabolism

Steven E. Raper

Key Points

1 Intermediary metabolic pathways – the metabolic manipulation and balancing of ingested

carbohydrate, fat, and protein – can process essentially all nutrients to acetyl coenzyme A (CoA) for

energy production predominantly through aerobic glycolysis, the citric acid cycle, and oxidative

phosphorylation.

2 Glucose must always be available for brain function; if not available directly from the diet, it can be

mobilized for a brief period from glycogen stores and then derived from proteins in the liver and

kidneys.

3 Free fatty acids are a direct source of energy for cardiac and skeletal muscles.

4 Hepatic protein synthesis, when excess amino acids are available, includes albumin, fibrinogen, and

apolipoproteins and can reach 50 g/day.

5 The citric acid cycle includes a series of mitochondrial enzymes that transform acetyl CoA – itself

derived from pyruvate or fatty acyl CoA – into water, carbon dioxide, and hydrogen-reducing

equivalents. Each molecule of acetyl CoA that enters the citric acid cycle yields 12 molecules of

adenosine triphosphate (ATP).

6 Oxidative phosphorylation converts the energy from NADH and FADH2

into ATP by the electron

transport chain and ATP synthase with a process called the proton motive force.

7 Biotransformation of potentially toxic, often hydrophobic, compounds into hydrophilic, excretable

compounds occurs mainly in the liver by the cytochromes P-450, the uridine diphosphate-glucuronyl

(UDP-glucuronyl) transferases, the glutathione (GSH) S-transferases, and the sulfotransferases.

INTERMEDIARY METABOLISM: AN OVERVIEW

Introduction

Intermediary metabolism – derived from the Greek word for change – is predominantly the fate of

dietary carbohydrate, fat, and protein in a series of life-sustaining cellular chemical transformations.

Although admittedly intricate, all surgeons should be familiar with the basics of the biochemistry by

which nutrients are converted to energy. Understanding the major biochemical pathways is a

prerequisite to making use of the rapid – and exciting – expansion of medical knowledge directed at

managing human metabolic derangement and improving health by beginning at the cellular level.

1 Intermediary metabolic pathways – the metabolic manipulation and balancing of ingested

carbohydrate, fat, and protein – can process essentially all nutrients to acetyl coenzyme A (CoA) for

energy production predominantly through aerobic glycolysis, the citric acid cycle, and oxidative

phosphorylation. The major intermediary metabolites are glucose, fatty acids, glycerol, and amino

acids. Glucose is metabolized to pyruvate and lactate by glycolysis. Aerobic metabolism allows

conversion of pyruvate to acetyl CoA. Acetyl CoA enters the citric acid cycle resulting in carbon dioxide,

water, and hydrogen-reducing equivalents (a major source of adenosine triphosphate [ATP]). In the

absence of oxygen, glycolysis ends in lactate. Glucose can be stored as or created from glycogen.

Glucose can also enter the phosphogluconate pathway, where it is converted to reducing equivalents for

fatty acid synthesis and ribose five-carbon sugars important in nucleotide formation. Glucose can be

converted into glycerol for fat formation and pyruvate for amino acid synthesis. Gluconeogenesis allows

synthesis of glucose from lactate, amino acids, and glycerol.

With regard to lipid metabolism, long-chain fatty acids arise from dietary fat or synthesized from

acetyl CoA. Fatty acids can be oxidized to acetyl CoA by the process of beta-oxidation or converted to

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acylglycerols (fat) for storage as the main energy reserve. In addition to the fats noted previously,

acetyl CoA can be used as a precursor to cholesterol and other steroids and in the liver can form the

ketone bodies, acetoacetate and 3-hydroxybutyrate, which are critical sources of energy during periods

of starvation.

Proteins are degraded in two major ways: an energy independent path, usually in lysosomes, and an

energy requiring path, usually through the ubiquitin pathway. About three-fourths of the amino acids

generated in protein catabolism are reutilized for protein synthesis and one-fourth is deaminated to

form ammonia and subsequently urea. Amino acids may be divided into nutritionally essential and

nonessential. Nonessential amino acids require fewer enzymatic reactions from amphibolic

intermediates or essential amino acids. Each day, humans turn over 1% to 2% of total body protein.

CARBOHYDRATE METABOLISM

The products of intestinal carbohydrate digestion are glucose (80%) and fructose and galactose (20%).

Fructose and galactose are rapidly converted to glucose, and the body uses glucose as the primary

molecule for transport and uptake of carbohydrates by cells throughout the body. Despite wide

fluctuations in dietary intake, blood glucose levels are tightly regulated by the liver. About 90% of

portal venous glucose is removed from the blood by liver cells through carrier-facilitated diffusion.

Large numbers of carrier molecules on the sinusoidal surface of the hepatocyte are capable of binding

glucose and transferring it to the cytoplasm. The rate of glucose transport is enhanced (up to 10-fold) by

insulin. Given the critical role of glucose in survival, complex metabolic pathways have evolved for the

storage of glucose in the fed state, the release of glucose from glycogen, and the synthesis of new

glucose.

Blood glucose is stored, primarily in liver and muscle, as glycogen. Glycogen is a complex polymer of

glucose with an average molecular weight of 5 million. The liver can convert up to 100 g of glucose

into glycogen per day by glycogenesis. The liver can also release glucose into the blood by

glycogenolysis, (breakdown of glycogen), or gluconeogenesis, (formation of new glucose from

substrates such as alanine, lactate, or glycerol). Hormones play a key role in the hepatic regulation of

glycogen balance. Insulin, for example, stimulates glycogenesis and glycolysis; glucagon stimulates

glycogenolysis and gluconeogenesis through cyclic adenosine monophosphate (AMP) and protein kinase

A.1

Glycogenesis and Glycogenolysis

2 Glucose must always be available for brain function; if not available directly from the diet, it can be

mobilized for a brief period from glycogen stores and then derived from proteins in the liver and

kidneys. The first step in glycogen storage is the transport of glucose through the plasma membrane.

Once in the hepatocyte, glucose and ATP are converted by the enzyme glucokinase to glucose-6-

phosphate (G6P), the first intermediate in the synthesis of glycogen (Fig. 2-1). Because complete

oxidation of one molecule of G6P generates 37 molecules of ATP, and storage uses only one molecule of

ATP, the overall efficiency of glucose storage as glycogen is a remarkable 97%.

Glycogenolysis does not occur by simple reversal of glycogenesis. Each glucose molecule on a

glycogen chain is released by glycogen phosphorylase (Fig. 2-2). Eventually, G6P is reformed. G6P

cannot exit from cells and must first be converted back to glucose. The conversion of G6P to glucose is

catalyzed by glucose-6-phosphatase, which exists only in hepatocytes, kidney, and intestinal epithelial

cells. Brain and muscle both use glucose as a primary fuel source and do not contain the phosphatase

enzyme. This lack of glucose-6-phosphatase ensures a ready supply of glucose for the energy needs of

brain and muscle. Liver uses glucose primarily as a precursor for other molecules and not for fuel.

Glycolysis

Glycolysis is the mammalian cellular pathway by which glucose is converted to pyruvate or lactate (Fig.

2-3). The glycolytic pathway is interesting in that glucose can be metabolized in the presence (aerobic)

or absence (anaerobic) of oxygen. Aerobic glycolysis is one of four stages in the oxidation of glucose

and the only stage that occurs in the cytosol. As will be discussed below, stages II to IV occur in

mitochondria; the citric acid cycle, electron transport generation of the proton motive force, and ATP

synthase leading to generation of ATP.

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Figure 2-1. The chemical reactions of glycogenesis and glycogenolysis. Glucose-6-phosphatase allows hepatic glucose to be

transported out of the hepatocyte for use in other tissues. Glucose-6-phosphate, in red, plays a central role in carbohydrate

metabolism.

Figure 2-2. Glucagon-stimulated enzyme cascade, responsible for the control of glycogen metabolism. Inactive forms are shown in

black, active forms in blue.

The aerobic conversion of glucose to pyruvate has three effects: (a) a net gain of two ATP molecules,

(b) generation of two reducing equivalents of the nicotinamide adenine nucleotide (NADH + H+), and

usually, (c) conversion of pyruvate to acetyl CoA with subsequent conversion of acetyl CoA in the

mitochondria to ATP. The conversion of glucose to pyruvate is regulated by three enzymes: hexokinase

(glucokinase), phosphofructokinase, and pyruvate kinase, which are nonequilibrium reactions and as

such, functionally irreversible.

Under anaerobic conditions, NADH cannot be reoxidized by transfer of reducing equivalents through

the electron transport chain to oxygen. Instead, pyruvate is reduced by NADH to lactate. Glycolysis

takes place in the cytoplasm, in contrast to the citric acid cycle and oxidative phosphorylation which are

mitochondrial processes. During times of glucose excess, as in the fed state, hepatic glycolysis can

generate energy in the form of ATP, but the oxidation of ketoacids is a preferred energy source in liver.

The conversion of lactate (through pyruvate) to glucose – a process possible only in the presence of

oxygen – is an important means of preventing severe lactic acidosis. Active skeletal muscles and

erythrocytes form large quantities of lactate. In patients with large wounds, lactate also accumulates.

The liver is exceptionally efficient at converting lactate to pyruvate through the Cori cycle (Fig. 2-4). As

a result, one would expect that only significant liver dysfunction would affect the Cori cycle and lead to

hyperlactatemia. However, lactate levels are now widely used to assess shock – septic and otherwise.2

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The hypothesis is that circulatory hypoperfusion impairs tissue oxygen delivery with resultant

mitochondrial hypoxia. In the absence of adequate oxygen, mitochondria switch to anaerobic glycolysis

and oxidative phosphorylation stops. As a result, serum lactate concentrations appear proportional to

ongoing tissue oxygenation deficits; thus improved lactate clearance can be used as a surrogate for

success of sepsis therapy.3 Serum lactate can also be used to assess prognosis and triage patients to ICU

level care.4

Figure 2-3. The glycolytic pathway. There is a net gain of two ATP molecules per glucose molecule. Phosphofructokinase is the

key regulatory enzyme in this pathway; however, all the enzymes in red catalyze irreversible reactions. The pathway shown here is

active only in the presence of aerobic conditions.

In erythrocytes, a unique variant of glycolysis enhances oxyhemoglobin dissociation. The first site in

glycolysis for generation of ATP is bypassed, leading to the formation of 2,3-bisphosphoglycerate by an

additional enzyme called bisphosphoglycerate mutase. Kinetics of the mutase present in erythrocytes

allow the presence of high concentrations of 2,3-bisphosphoglycerate to build up. The 2,3-

bisphosphoglycerate displaces oxygen from hemoglobin, allowing a shift of the oxyhemoglobin

dissociation curve to the right.

Gluconeogenesis

There is an absolute minimum requirement for glucose in humans. Below a certain blood glucose

concentration, brain dysfunction causes coma and death. When glucose becomes scarce, as in the fasting

state, glycogenolysis occurs. Once glycogen stores have been depleted, the liver and kidneys are capable

of synthesizing new glucose by the process of gluconeogenesis. Glucagon is produced in response to low

blood sugar levels and stimulates gluconeogenesis.

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Figure 2-4. The gluconeogenesis pathway. The irreversible nature of the glycolytic pathway means that a different sequence of

biosyntheses is required for glucose production. The enzymes in red catalyze irreversible reactions that are different from those in

glycolysis. In mammals, glucose cannot be synthesized from acetyl coenzyme A, only from cytosolic pyruvate.

Gluconeogenesis is not a simple reversal of the glycolytic pathway. In glycolysis, as noted previously,

the conversion of glucose to pyruvate is a one-way reaction. As a result, four separate, functionally

irreversible enzyme reactions are required to convert pyruvate into glucose (Fig. 2-5). These enzymes

are pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and

glucose-6-phosphatase. Other enzymes are shared with the glycolytic pathway.

About 60% of the naturally occurring amino acids, glycerol, or lactate can also be used as substrates

for glucose production. Alanine is the amino acid most easily converted into glucose. Simple

deamination allows conversion to pyruvate, which is subsequently converted to glucose. Other amino

acids can be converted into three-, four-, or five-carbon sugars and then enter the phosphogluconate

pathway (next section). Gluconeogenesis is enhanced by fasting, critical illness, and periods of anaerobic

metabolism.

Figure 2-5. The Cori cycle, an elegant mechanism for the hepatic conversion of muscle lactate into new glucose. Pyruvate plays a

key role in this process.

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Phosphogluconate Pathway

When glucose enters the liver, glycogen is formed until the hepatic glycogen capacity is reached (about

100 g). If excess glucose is still available, the liver converts it to fat by the phosphogluconate pathway

(also known as the pentose phosphate pathway) (Fig. 2-6). The cytosolic phosphogluconate pathway can

completely oxidize glucose, generating CO2 and nicotinamide adenine dinucleotide phosphate (NADPH)

through what is known as the oxidative phase. Hydrogen atoms released in the phosphogluconate

pathway combine with oxidized nicotinamide adenine dinucleotide phosphate (NADP+) to form reduced

nicotinamide adenine dinucleotide phosphate (NADPH − H+).5 The oxidative phase is present only in

tissues, such as the adrenal glands and gonads, that require reductive biosyntheses such as

steroidogenesis or other forms of lipid synthesis. Essentially, all tissues contain the nonoxidative phase,

which is reversible and produces ribose precursors for nucleotide synthesis. In erythrocytes, the

phosphogluconate pathway provides reducing equivalents for the production of reduced glutathione by

glutathione reductase. Reduced glutathione can remove hydrogen peroxide, which increases the

conversion of oxyhemoglobin to methemoglobin and subsequent hemolysis.

LIPID METABOLISM

Lipid Transport

Lipid transport throughout the body is made complicated by the fact that lipids are insoluble in water.

To overcome this physicochemical incompatibility, dietary triglycerides are first split into

monoglycerides and fatty acids by the action of intestinal lipases. After absorption into small intestinal

cells, triacylglycerols are reformed and aggregate into chylomicrons, which then enter the bloodstream

by way of lymph. Chylomicrons are removed from the blood by the liver and adipose tissue. The

capillary surface of the liver contains large amounts of lipoprotein lipase, which hydrolyzes triglycerides

into fatty acids and glycerol. The fatty acids freely diffuse into hepatocytes for further metabolism.

Similar to chylomicrons, very low-density lipoproteins (VLDLs) are synthesized by the liver and are the

main vehicle for transport of triacylglycerols to extrahepatic tissues. The intestines and liver are the

only two tissues capable of secreting lipid particles. In addition to chylomicrons and VLDLs, there are

two other major groups of plasma lipoproteins: low-density lipoproteins (LDLs) and high-density

lipoproteins (HDLs). LDLs and HDLs contain predominantly cholesterol and phospholipid.

Figure 2-6. The phosphogluconate pathway. One of the major purposes of this pathway is to generate reduced nicotinamide

adenine dinucleotide, which can serve as an electron donor and allow the liver to perform reductive biosynthesis. Glucose-6-

phosphate, in red, plays a central role in carbohydrate metabolism.

The structure of all classes of lipoproteins is similar. There is a core of nonpolar lipids, either

triacylglycerols or cholesteryl esters, depending on the particular lipoprotein. This nonpolar core is

coated with a surface layer of amphipathic phospholipid or cholesterol oriented so that the polar ends

are in contact with the plasma. A protein component is also present. The A apolipoproteins occur in

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chylomicrons and HDLs. The B apolipoproteins come in two forms: B-100 is the predominant

apolipoprotein of LDLs, whereas the shorter B-48 is located in chylomicrons. The C apolipoproteins can

transfer between VLDLs, LDLs, and HDLs. Apolipoproteins D and E also exist. Apolipoproteins have

several functions in lipid transport and storage. Some, such as the B apolipoproteins, are an integral part

of the lipoprotein structure. Other apolipoproteins are enzyme cofactors, such as C-II for lipoprotein

lipase. Lastly, the apolipoproteins act as ligands for cell surface receptors. As an example, both B-100

and E serve as ligands for the LDL receptor.6

Plasma variations in LDL cholesterol, HDL cholesterol, and triglycerides affect risk for atherosclerotic

cardiovascular disease. As dyslipidemias are being identified and studied, new therapeutic approaches

are needed. A convergence of human genetics and functional biology has led to recent advances in the

study of lipoprotein metabolism. Genome-wide association studies have identified about 100 genes

associated with plasma lipid phenotypes – many of which were not previously known to be associated

with lipids. These genes are now being functionally validated through human genetic analysis such as

deep targeted resequencing of kindreds with Mendelian lipid abnormalities or gene manipulation (overor underexpression) in cultured cells and animal models.7

FATTY ACID METABOLISM

Most human fatty acids in plasma are long-chain acids (C-16 to C-20). Because long-chain fatty acids are

not readily absorbed by the intestinal mucosa, they must first be incorporated into chylomicrons. In

contrast, short-chain and medium-chain fatty acids are absorbed directly into the portal circulation and

are avidly taken up by the liver. Free fatty acids in the circulation are noncovalently bound to albumin

and are transferred to the hepatocyte cytosol by way of fatty acid–binding proteins. Fatty acid-CoA

esters are synthesized in the cytosol after hepatic uptake of fatty acids. These fatty acid-CoA esters can

be converted into triglyceride, transported into mitochondria for the production of acetyl CoA and

reducing equivalents, or stored in the liver as triglycerides. The rate-limiting step in the synthesis of

triglyceride is the conversion of acetyl CoA to malonyl CoA. Malonyl CoA, in turn, inhibits the

mitochondrial uptake of fatty acid-CoA ester, favoring triglyceride synthesis. The liver also contains

dehydrogenases that can unsaturate essential dietary fatty acids. Structural elements of all tissues

contain significant amounts of unsaturated fats, and the liver is responsible for the production of these

unsaturated fatty acids. As another example, dietary linoleic acid is elongated and dehydrogenated to

the prostaglandin precursor arachidonic acid.

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