17.4 What Experiments Can Be Used to Elucidate Metabolic Pathways? 523 NADPH Provides the Reducing Power for Anabolic Processes Whereas catabolism is fundamentally an oxidative process, anabolism is, by its con- trasting nature, reductive. The biosynthesis of the complex constituents of the cell begins at the level of intermediates derived from the degradative pathways of catab- olism; or, less commonly, biosynthesis begins with oxidized substances available in the inanimate environment, such as carbon dioxide. When the hydrocarbon chains of fatty acids are assembled from acetyl-CoA units, activated hydrogens are needed to reduce the carbonyl (CPO) carbon of acetyl-CoA into a OCH 2 O at every other position along the chain. When glucose is synthesized from CO 2 during photosyn- thesis in plants, reducing power is required. These reducing equivalents are pro- vided by NADPH, the usual source of high-energy hydrogens for reductive biosyn- thesis. NADPH is generated when NADP ϩ is reduced with electrons in the form of hydride ions. In heterotrophic organisms, these electrons are removed from fuel molecules by NADP ϩ -specific dehydrogenases. In these organisms, NADPH can be viewed as the carrier of electrons from catabolic reactions to anabolic reactions (Figure 17.12). In photosynthetic organisms, the energy of light is used to pull elec- trons from water and transfer them to NADP ϩ ; O 2 is a by-product of this process. Coenzymes and Vitamins Provide Unique Chemistry and Essential Nutrients to Pathways In addition to NAD ϩ and NADPH, a variety of other small molecules are essential to metabolism. Some of these are essential nutrients called vitamins. (The name was coined by Kazimierz Funk, who discovered thiamine as a cure for beriberi in 1912 and termed it a “vital amine.” He later proposed that other diseases might be cured by “vitamins.”) Vitamins are required in the diet, usually in trace amounts, because they cannot be synthesized by the organism itself. The requirement for any given vitamin de- pends on the organism. Not all vitamins are required by all organisms. Vitamins re- quired in the human diet are listed in Table 17.3. These important substances are traditionally distinguished as being either water soluble or fat soluble. Except for vitamin C (ascorbic acid), the water-soluble vitamins are all compo- nents or precursors of important biological substances known as coenzymes. These are low-molecular-weight molecules that bring unique chemical functionality to certain enzyme reactions. Coenzymes may also act as carriers of specific functional groups, such as methyl groups and acyl groups. The side chains of the common amino acids provide only a limited range of chemical reactivities and carrier prop- erties. Coenzymes, acting in concert with appropriate enzymes, provide a broader range of catalytic properties for the reactions of metabolism. Coenzymes are typi- cally modified by these reactions and are then converted back to their original forms by other enzymes, so small amounts of these substances can be used repeat- edly. The coenzymes derived from the water-soluble vitamins are listed in Table 17.3. Each will be discussed in the context of the chemistry they provide to specific pathways in Chapters 18 through 27. The fat-soluble vitamins are not directly re- lated to coenzymes, but they play essential roles in a variety of critical biological processes, including vision, maintenance of bone structure, and blood coagula- tion. The mechanisms of action of fat-soluble vitamins are not as well understood as their water-soluble counterparts, but modern research efforts are gradually clos- ing this gap. 17.4 What Experiments Can Be Used to Elucidate Metabolic Pathways? Armed with the knowledge that metabolism is organized into pathways of successive reactions, we can appreciate by hindsight the techniques employed by early bio- chemists to reveal their sequence. A major intellectual advance took place at the Catabolism Reductive biosynthetic reactions Reductive biosynthetic product Oxidized precursor Reduced fuel Oxidized product NADP + NADPH FIGURE 17.12 Transfer of reducing equivalents from catabolism to anabolism via the NADPH cycle. 524 Chapter 17 Metabolism: An Overview end of the 19th century when Eduard Buchner showed that the fermentation of glu- cose to yield ethanol and carbon dioxide can occur in extracts of broken yeast cells. Until this discovery, many thought that metabolism was a vital property, unique to intact cells; even the eminent microbiologist Louis Pasteur, who contributed so much to our understanding of fermentation, was a vitalist, one of those who be- lieved that the processes of living substance transcend the laws of chemistry and physics. After Buchner’s revelation, biochemists searched for intermediates in the transformation of glucose and soon learned that inorganic phosphate was essential to glucose breakdown. This observation gradually led to the discovery of a variety of phosphorylated organic compounds that serve as intermediates along the fermen- tative pathway. An important tool for elucidating the steps in the pathway was the use of meta- bolic inhibitors. Adding an enzyme inhibitor to a cell-free extract caused an accumu- lation of intermediates in the pathway prior to the point of inhibition (Figure 17.13). Each inhibitor was specific for a particular site in the sequence of metabolic events. As the arsenal of inhibitors was expanded, the individual steps in metabo- lism were revealed. Discussed Vitamin Coenzyme Form Function in Chapter Water-Soluble Thiamine (vitamin B 1 ) Niacin (nicotinic acid) Riboflavin (vitamin B 2 ) Pantothenic acid Pyridoxal, pyridoxine, pyridoxamine (vitamin B 6 ) Cobalamin (vitamin B 12 ) Biotin Lipoic acid Folic acid Fat-Soluble Retinol (vitamin A) Retinal (vitamin A) Retinoic acid (vitamin A) Ergocalciferol (vitamin D 2 ) Cholecalciferol (vitamin D 3 ) ␣-Tocopherol (vitamin E) Menaquinone (vitamin K) TABLE 17.3 Vitamins and Coenzymes Thiamine pyrophosphate Nicotinamide adenine dinucleotide (NAD ϩ ) Nicotinamide adenine dinucleo- tide phosphate (NADP ϩ ) Flavin adenine dinucleotide (FAD) Flavin mononucleotide (FMN) Coenzyme A Pyridoxal phosphate 5Ј-Deoxyadenosylcobalamin Methylcobalamin Biotin–lysine complexes (biocytin) Lipoyl–lysine complexes (lipoamide) Tetrahydrofolate Decarboxylation of ␣-keto acids and formation and cleavage of ␣-hydroxyketones Hydride transfer Hydride transfer One- and two-electron transfer One- and two-electron transfer Activation of acyl groups for transfer by nucleophilic attack, and activation of the ␣-hydrogen of the acyl group for abstraction as a proton Formation of stable Schiff base (aldimine) adducts with ␣-amino groups of amino acids; serving as an electron sink to stabilize reaction intermediates Intramolecular rearrangement, reduction of ribonucleotides to deoxyribonucleotides, and methyl group transfer Carrier of carboxyl groups in carboxylation reactions Coupling acyl group transfer and electron transfer during oxidation and decarboxylation of ␣-keto acids Acceptor and donor of 1-C units for all oxidation levels of carbon except that of CO 2 19, 22 18–27 21, 22, 24–26 19, 20, 23, 26 20 19, 23, 24, 27 25 23 22, 24 19 25, 26 17.4 What Experiments Can Be Used to Elucidate Metabolic Pathways? 525 E 1 E 2 E 3 E 4 E 5 E 6 E 1 E 2 E 3 E 4 E 5 E 6 Metabolite concentration Substrate B C D E F Product Control: BCDEF Intermediate Metabolite concentration Substrate B C D E F Produc t Plus inhibitor, I, of E 4 : BCDEF Inhibitor Intermediate (b) (a) FIGURE 17.13 The use of inhibitors to reveal the se- quence of reactions in a metabolic pathway. (a) Control: Under normal conditions, the steady-state concentra- tions of a series of intermediates will be determined by the relative activities of the enzymes in the pathway. (b) Plus inhibitor: In the presence of an inhibitor (in this case, an inhibitor of enzyme 4 ), intermediates upstream of the metabolic block (B, C, and D) accumulate, reveal- ing themselves as intermediates in the pathway.The concentration of intermediates lying downstream (E and F) will fall. Isotope Type Radiation Type Half-Life Relative Abundance* 2 H Stable 0.0154% 3 H Radioactive  Ϫ 12.1 years 13 C Stable 1.1% 14 C Radioactive  Ϫ 5700 years 15 N Stable 0.365% 18 O Stable 0.204% 24 Na Radioactive  Ϫ , ␥ 15 hours 32 P Radioactive  Ϫ 14.3 days 35 S Radioactive  Ϫ 87.1 days 36 Cl Radioactive  Ϫ 310,000 years 42 K Radioactive  Ϫ 12.5 hours 45 Ca Radioactive  Ϫ 152 days 59 Fe Radioactive  Ϫ , ␥ 45 days 131 I Radioactive  Ϫ , ␥ 8 days *The relative natural abundance of a stable isotope is important because, in tracer studies, the amount of stable isotope is typically expressed in terms of atoms percent excess over the natural abundance of the isotope. TABLE 17.4 Properties of Radioactive and Stable “Heavy” Isotopes Used as Tracers in Metabolic Studies Mutations Create Specific Metabolic Blocks Genetics provides an approach to the identification of intermediate steps in me- tabolism that is somewhat analogous to inhibition. Mutation in a gene encoding an enzyme often results in an inability to synthesize the enzyme in an active form. Such a defect leads to a block in the metabolic pathway at the point where the enzyme acts, and the enzyme’s substrate accumulates. Such genetic disorders are lethal if the end product of the pathway is essential or if the accumulated intermediates have toxic effects. In microorganisms, however, it is often possible to manipulate the growth medium so that essential end products are provided. Then the biochemical consequences of the mutation can be investigated. Studies on mutations in genes of the filamentous fungus Neurospora crassa led G. W. Beadle and E. L. Tatum to hy- pothesize in 1941 that genes are units of heredity that encode enzymes (a principle referred to as the “one gene–one enzyme” hypothesis). Isotopic Tracers Can Be Used as Metabolic Probes Another widely used approach to the elucidation of metabolic sequences is to “feed” cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard: radioac- tive isotopes, such as 14 C, and stable “heavy” isotopes, such as 18 O or 15 N (Table 17.4). 526 Chapter 17 Metabolism: An Overview Because the chemical behavior of isotopically labeled compounds is rarely distin- guishable from that of their unlabeled counterparts, isotopes provide reliable “tags” for observing metabolic changes. The metabolic fate of a radioactively labeled sub- stance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 17.14). Heavy Isotopes Heavy isotopes endow the compounds in which they appear with slightly greater masses than their unlabeled counterparts. These compounds can be separated and quantitated by mass spectrometry (or density gradient centrifuga- tion, if they are macromolecules). For example, 18 O was used in separate experi- ments as a tracer of the fate of the oxygen atoms in water and carbon dioxide to de- termine whether the atmospheric oxygen produced in photosynthesis arose from H 2 O, CO 2 , or both: CO 2 ϩ H 2 O ⎯⎯→ (CH 2 O) ϩ O 2 If 18 O-labeled CO 2 was presented to a green plant carrying out photosynthesis, none of the 18 O was found in O 2 . Curiously, it was recovered as H 2 18 O. In contrast, when plants fixing CO 2 were equilibrated with H 2 18 O, 18 O 2 was evolved. These lat- ter labeling experiments established that photosynthesis is best described by the equation C 16 O 2 ϩ 2 H 2 18 O ⎯⎯→ (CH 2 16 O) ϩ 18 O 2 ϩ H 2 16 O That is, in the process of photosynthesis, the two oxygen atoms in O 2 come from two H 2 O molecules. One O is lost from CO 2 and appears in H 2 O, and the other O of CO 2 is retained in the carbohydrate product. Two of the four H atoms are accounted for in (CH 2 O), and two reduce the O lost from CO 2 to H 2 O. NMR Spectroscopy Is a Noninvasive Metabolic Probe A technology analogous to isotopic tracers is provided by nuclear magnetic resonance (NMR) spectroscopy. The atomic nuclei of certain isotopes, such as the naturally occurring isotope of phosphorus, 31 P, have magnetic moments. The reso- nance frequency of a magnetic moment is influenced by the local chemical envi- ronment. That is, the NMR signal of the nucleus is influenced in an identifiable way by the chemical nature of its neighboring atoms in the compound. In many ways, these nuclei are ideal tracers because their signals contain a great deal of structural information about the environment around the atom and thus the FIGURE 17.14 One of the earliest experiments using a ra- dioactive isotope as a metabolic tracer. Cells of Chlorella (a green alga) synthesizing carbohydrate from carbon dioxide were exposed briefly (5 sec) to 14 C-labeled CO 2 . The products of CO 2 incorporation were then quickly iso- lated from the cells, separated by two-dimensional paper chromatography, and observed via autoradiographic ex- posure of the chromatogram. Such experiments identi- fied radioactive 3-phosphoglycerate (PGA) as the pri- mary product of CO 2 fixation.The 3-phosphoglycerate was labeled in the 1-position (in its carboxyl group). Ra- dioactive compounds arising from the conversion of 3-phosphoglycerate to other metabolic intermediates included phosphoenolpyruvate (PEP), malic acid, triose phosphate, alanine, and sugar phosphates and diphosphates. Courtesy of Professor Melvin Calvin, Lawrence Berkeley Laboratory , University of California, Berkeley 17.4 What Experiments Can Be Used to Elucidate Metabolic Pathways? 527 nature of the compound containing the atom. Transformations of substrates and metabolic intermediates labeled with magnetic nuclei can be traced by following changes in NMR spectra. Furthermore, NMR spectroscopy is a noninvasive proce- dure. Whole-body NMR spectrometers are being used today in hospitals to directly observe the metabolism (and clinical condition) of living subjects (Figure 17.15). NMR promises to be a revolutionary tool for clinical diagnosis and for the investi- gation of metabolism in situ (literally “in site,” meaning, in this case, “where and as it happens”). Metabolic Pathways Are Compartmentalized Within Cells Although the interior of a prokaryotic cell is not subdivided into compartments by internal membranes, the cell still shows some segregation of metabolism. For ex- ample, certain metabolic pathways, such as phospholipid synthesis and oxidative phosphorylation, are localized in the plasma membrane. Protein biosynthesis is car- ried out on ribosomes. In contrast, eukaryotic cells are extensively compartmentalized by an endo- membrane system. Each of these cells has a true nucleus bounded by a double membrane called the nuclear envelope. The nuclear envelope is continuous with the endomembrane system, which is composed of differentiated regions: the en- doplasmic reticulum; the Golgi complex; various membrane-bounded vesicles such as lysosomes, vacuoles, and microbodies; and, ultimately, the plasma mem- brane itself. Eukaryotic cells also possess mitochondria and, if they are photo- synthetic, chloroplasts. Disruption of the cell membrane and fractionation of the cell contents into the component organelles have allowed an analysis of their re- spective functions (Figure 17.16). Each compartment is dedicated to specialized metabolic functions, and the enzymes appropriate to these specialized functions are confined together within the organelle. In many instances, the enzymes of a metabolic sequence occur together within the organellar membrane. Thus, the flow of metabolic intermediates in the cell is spatially as well as chemically segregated. For example, the 10 enzymes of glycolysis are found in the cytosol, but pyruvate, the product of glycolysis, is fed into the mitochondria. These organelles contain the citric acid cycle enzymes, which oxidize pyruvate to CO 2 . The great amount of energy released in the process is captured by the oxidative phosphorylation system of mitochondrial membranes and used to drive the formation of ATP (Figure 17.17). (a) ?Strength of 31 P signal 10 0 –10 –20 ppm Chemical shift Before exercise Phosphocreatine ATP ␥  ␣ P i 10 0 –10 –20 ppm Chemical shift (b) During exercise P i Phosphocreatine ␥  ␣ ?Strength of 31 P signal FIGURE 17.15 With NMR spectroscopy, one can observe the metabolism of a living subject in real time.These NMR spectra show the changes in ATP, creatine-P (phos- phocreatine), and P i levels in the forearm muscle of a human subjected to 19 minutes of exercise. Note that the three P atoms of ATP (␣, , and ␥) have different chemical shifts, reflecting their different chemical environments. 528 Chapter 17 Metabolism: An Overview 600 rpm Teflon pestle Tissue–sucrose homogenate (minced tissue + 0.25 M sucrose buffer) Strain homogenate to remove connective tissue and blood vessels. Tube is moved slowly up and down as pestle rotates. Centrifuge homogenate at 600 g × 10 min. Centrifuge supernatant 1 at 15,000 g × 5 min. Supernatant 1 Supernatant 2 Centrifuge supernatant 2 at 100,000 g × 60 min. Nuclei and any unbroken cells Mitochondria, lysosomes, and microbodies Supernatant 3: Soluble fraction of cytoplasm (cytosol) Ribosomes and microsomes, consisting of endoplasmic reticulum, Golgi, and plasma membrane fragments FIGURE 17.16 Fractionation of a cell extract by differential centrifugation. It is possible to separate organelles and subcellular particles in a centrifuge because their inherent size and density differences give them different rates of sedimentation in an applied centrifugal field. Nuclei are pelleted in relatively weak centrifugal fields and mitochondria in somewhat stronger fields, whereas very strong centrifugal fields are necessary to pellet ribo- somes and fragments of the endomembrane system. 17.5 What Can the Metabolome Tell Us about a Biological System? 529 17.5 What Can the Metabolome Tell Us about a Biological System? Rapid advances in chemical analysis have made it possible to carry out comprehen- sive studies of the many metabolites in a living organism. The metabolome is the complete set of low-molecular-weight molecules present in an organism or excreted by it under a given set of physiological conditions. Metabolomics is the systematic identification and quantitation of all these metabolites in a given organism or sam- ple. It is quite remarkable that biochemists can foresee the rise of a true systems biology, where comprehensive information sets from the genome, the transcrip- tome, the proteome, and the metabolome will combine to provide incisive descrip- tions of biological systems and detailed understanding of many human diseases. Even simple organisms present daunting challenges for metabolomic analyses. There are more than 500 metabolites represented in Figure 17.2, but far more ex- ist in a typical cell. For example, the 40 or so fatty acids occurring in a cell can alone account for thousands of different metabolites. (Triglycerides, with three fatty acids esterified to a glycerol backbone, could account for 40 ϫ 40 ϫ 40 ϭ 64,000 species by themselves!) The Human Metabolomics Database (www.hmdb.ca) provides data on more than 2500 metabolites known in cells of the human body and human body fluids (blood, urine, and so on). Metabolomic measurements must be able to re- solve and discriminate this array of small molecules. Moreover, concentrations of metabolites vary widely, from 10 Ϫ12 M (for many hormones) to 0.1 M (for Na ϩ ions). Comprehensive metabolomic analyses involve processing of many samples, so the time and cost required per sample must be as low as possible. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) are both pow- erful techniques for metabolomic analysis. Mass spectrometry offers unmatched sen- sitivity for detection of metabolites at low concentrations (Figure 17.18), and NMR spectroscopy can provide remarkable resolution and discrimination of metabolites in complex mixtures (Figure 17.19). Combination of these techniques with a variety of chromatographic separation protocols (Figure 17.20) makes it possible to analyze thousands of metabolites in biological samples rapidly and at low cost. 100 50 Relative abundance 0 80 160 240 m/z 320 Control 100 50 Relative abundance 0 80 160 240 m/z 320 PKU 100 50 Relative abundance 0 80 160 240 m/z 320 HCY 100 50 Relative abundance 0 80 160 240 m/z 320 MSUD FIGURE 17.18 Mass spectrometry offers remarkable sensitivity for metabolomic analyses. Shown here are desorption electrospray ionization mass spectra for urine samples from individuals with inborn errors of metabolism. PKU ϭ phenylketonuria; HCY ϭ homo- cystinuria; MSUD ϭ maple syrup urine disease. (Adapted from Pan, Z., Gu, H., et al., 2007. Principal component analysis of urine metabolites by NMR and DESI-MS in patients with inborn errors of metabolism. Analytical and Bioanalytical Chemistry 387:539–549.) ATP 2 + Glucose Glucose 2 Pyruvate Glycolysis in the cytosol Acetyl-CoA Citric acid cycle Citric acid cycle and oxidative phosphoryla- tion in the mitochondria ATP ATP ATP NADH CO 2 O 2 2 NADH NAD + NADH ADP H 2 O P i ATP 2 FIGURE 17.17 Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation. 530 Chapter 17 Metabolism: An Overview 20 13 C (ppm) 80 60 40 54321 Arabidopsis extract Mixture of standards 1 H (ppm) (a) 1 H NMR (b) 1 H- 13 C NMR FIGURE 17.19 (a) One-dimensional 1 H NMR spectrum of an equimolar mixture of 26 small-molecule standards. (b) Two- dimensional NMR spectrum of the same mixture (red) overlaid onto a spectrum of aqueous whole-plant extract from Arabidopsis thaliana, a model organism for the study of plant molecular biology and biochemistry. (Adapted from Lewis, I., Schommer, S., et al., 2007. Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1 H- 13 C NMR spectra. Analytical Chemistry 79:9385–9390.) 100 Relative intensity (% base peak) 0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 Time FIGURE 17.20 The combination of mass spectrometry and gas chromatography makes it possible to separate and identify hundreds of metabolites. Shown is an ion chromatogram of a human urine sample, with 1582 separately identified peaks. (Adapted from Dettmer, K., and Aronov, P., 2007. Mass spectrometry-based metabolomics. Mass Spectrometry Reviews 26:51–79.) 17.6 What Food Substances Form the Basis of Human Nutrition? 531 17.6 What Food Substances Form the Basis of Human Nutrition? The use of foods by organisms is termed nutrition. The ability of an organism to use a particular food material depends upon its chemical composition and upon the meta- bolic pathways available to the organism. In addition to essential fiber, food includes the macronutrients—protein, carbohydrate, and lipid—and the micronutrients— including vitamins and minerals. Humans Require Protein Humans must consume protein in order to make new proteins. Dietary protein is a rich source of nitrogen, and certain amino acids—the so-called essential amino acids—cannot be synthesized by humans (and various animals) and can be ob- tained only in the diet. The average adult in the United States consumes far more protein than required for synthesis of essential proteins. Excess dietary protein is then merely a source of metabolic energy. Some of the amino acids (termed gluco- genic) can be converted into glucose, whereas others, the ketogenic amino acids, can be converted to fatty acids and/or keto acids. If fat and carbohydrate are al- ready adequate for the energy needs of the individual, then both kinds of amino acids will be converted to triacylglycerol and stored in adipose tissue. An individual’s protein undergoes a constant process of degradation and resyn- thesis. Together with dietary protein, this recycled protein material participates in a nitrogen equilibrium, or nitrogen balance. A positive nitrogen balance occurs whenever there is a net increase in the organism’s protein content, such as during periods of growth. A negative nitrogen balance exists when dietary intake of nitrogen is insufficient to meet the demands for new protein synthesis. Carbohydrates Provide Metabolic Energy The principal purpose of carbohydrate in the diet is production of metabolic energy. Simple sugars are metabolized in the glycolytic pathway (see Chapter 18). Complex carbohydrates are degraded into simple sugars, which then can enter the glycolytic pathway. Carbohydrates are also essential components of nucleotides, nucleic acids, glycoproteins, and glycolipids. Human metabolism can adapt to a wide range of dietary carbohydrate levels, but the brain requires glucose for fuel. When di- etary carbohydrate consumption exceeds the energy needs of the individual, ex- cess carbohydrate is converted to triacylglycerols and glycogen for long-term en- ergy storage. On the other hand, when dietary carbohydrate intake is low, ketone bodies are formed from acetate units to provide metabolic fuel for the brain and other organs. Lipids Are Essential, But in Moderation Fatty acids and triacylglycerols can be used as fuel by many tissues in the human body, and phospholipids are essential components of all biological membranes. Even though the human body can tolerate a wide range of fat intake levels, there are disadvantages in either extreme. Excess dietary fat is stored as triacylglycerols in adipose tissue, but high levels of dietary fat can also increase the risk of athero- sclerosis and heart disease. Moreover, high dietary fat levels are also correlated with increased risk for colon, breast, and prostate cancers. When dietary fat consump- tion is low, there is a risk of essential fatty acid deficiencies. As will be seen in Chap- ter 24, the human body cannot synthesize linoleic and linolenic acids, so these must be acquired in the diet. In addition, arachidonic acid can by synthesized in humans only from linoleic acid, so it too is classified as essential. The essential fatty acids are key components of biological membranes, and arachidonic acid is the precursor to prostaglandins, which mediate a variety of processes in the body. 532 Chapter 17 Metabolism: An Overview Fiber May Be Soluble or Insoluble The components of food materials that cannot be broken down by human digestive enzymes are referred to as dietary fiber. There are several kinds of dietary fiber, each with its own chemical and biological properties. Cellulose and hemicellulose are in- soluble fiber materials that stimulate regular function of the colon. They may play a role in reducing the risk of colon cancer. Lignins, another class of insoluble fibers, ab- sorb organic molecules in the digestive system. Lignins bind cholesterol and clear it from the digestive system, reducing the risk of heart disease. Pectins and gums are wa- ter-soluble fiber materials that form viscous gel-like suspensions in the digestive system, slowing the rate of absorption of many nutrients, including carbohydrates, and lower- ing serum cholesterol in many cases. The insoluble fibers are prevalent in vegetable grains. Water-soluble fiber is a component of fruits, legumes, and oats. A DEEPER LOOK A Popular Fad Diet—Low Carbohydrates, High Protein, High Fat Possibly the most serious nutrition problem in the United States is excessive food consumption, and many people have experimented with fad diets in the hope of losing excess weight. One of the most popular of the fad diets has been the high-protein, high-fat (low- carbohydrate) diet. The presumed rationale is tantalizing: Be- cause the tricarboxylic acid (TCA) cycle (see Chapter 19) plays a key role in fat catabolism and because glucose is usually needed to replenish intermediates in the TCA cycle, if carbohydrates are re- stricted in the diet, dietary fat should merely be converted to ke- tone bodies and excreted. This so-called diet appears to work at first because a low-carbohydrate diet results in an initial water (and weight) loss. This occurs because glycogen reserves are de- pleted by the diet and because about 3 grams of water of hydration are lost for every gram of glycogen. However, the premise for such diets is erroneous for several reasons. First, ketone body excretion by the human body usually does not exceed 20 grams (400 kJ) per day. Second, amino acids can function effectively to replenish TCA cycle intermediates, making the reduced carbohydrate regimen irrelevant. Third, the typical fare in a high-protein, high-fat, low-carbohydrate diet is expensive but not very tasty, and it is thus difficult to maintain. Finally, a diet high in saturated and trans fatty acids is a high risk factor for atherosclerosis and coronary artery disease. SUMMARY 17.1 Is Metabolism Similar in Different Organisms? One of the great unifying principles of modern biology is that organisms show marked similarity in their major pathways of metabolism. Given the almost un- limited possibilities within organic chemistry, this generality would ap- pear most unlikely. Yet it’s true, and it provides strong evidence that all life has descended from a common ancestral form. All forms of nutri- tion and almost all metabolic pathways evolved in early prokaryotes prior to the appearance of eukaryotes 1 billion years ago. All organisms, even those that can synthesize their own glucose, are capable of glucose degradation and ATP synthesis via glycolysis. Other prominent pathways are also virtually ubiquitous among organisms. 17.2 What Can Be Learned from Metabolic Maps? Metabolism repre- sents the sum of the chemical changes that convert nutrients, the “raw materials” necessary to sustain living organisms, into energy and the chemically complex finished products of cells. Metabolism consists of literally hundreds of enzymatic reactions organized into discrete path- ways. Metabolic maps portray the principal reactions of the intermedi- ary metabolism of carbohydrates, lipids, amino acids, and their deriva- tives. In such maps, arrows connect metabolites and represent the enzyme reactions that interconvert the metabolites. Alternative map- pings of biochemical pathways have been proposed in a response to the emergence of genomic, transcriptomic, and proteomic perspectives on the complexity of biological systems. 17.3 How Do Anabolic and Catabolic Processes Form the Core of Meta- bolic Pathways? Catabolism involves the oxidative degradation of com- plex nutrient molecules (carbohydrates, lipids, and proteins) obtained either from the environment or from cellular reserves. The breakdown of these molecules by catabolism leads to the formation of simpler mol- ecules such as lactic acid, ethanol, carbon dioxide, urea, or ammonia. Catabolic reactions are usually exergonic, and often the chemical en- ergy released is captured in the form of ATP. Anabolism is a synthetic process in which the varied and complex biomolecules (proteins, nu- cleic acids, polysaccharides, and lipids) are assembled from simpler pre- cursors. Such biosynthesis involves the formation of new covalent bonds, and an input of chemical energy is necessary to drive such en- dergonic processes. The ATP generated by catabolism provides this en- ergy. Furthermore, NADPH is an excellent donor of high-energy elec- trons for the reductive reactions of anabolism. 17.4 What Experiments Can Be Used to Elucidate Metabolic Pathways? An important tool for elucidating the steps in the pathway is the use of metabolic inhibitors. Adding an enzyme inhibitor to a cell-free extract causes an accumulation of intermediates in the pathway prior to the point of inhibition. Each inhibitor is specific for a particular site in the sequence of metabolic events. Genetics provides an approach to the identification of intermediate steps in metabolism that is somewhat analogous to inhibition. Mutation in a gene encoding an enzyme often results in an inability to synthesize the enzyme in an active form. Such a defect leads to a block in the metabolic pathway at the point where the enzyme acts, and the enzyme’s substrate accumulates. Such genetic dis- orders are lethal if the end product of the pathway is essential or if the accumulated intermediates have toxic effects. In microorganisms, how- ever, it is often possible to manipulate the growth medium so that essen-