CHAPTER Be OBJECTIVES 1. Define the folowing tr: Cartotyerate onossccherde, saccharide, and polysaccharide Ketone Insulin Diabetes mettus iycogen lycogenesis lycogenalysis Biyeotsis luconeogenesss ‘ucose tolerance Hyperglycemia and hypoglycemia Gycation 2. Provide examples ofa monosaccharide, disaccharide, and Polysaccharide 3, Discus the reguaton of glucose concentration in the boxy and state ‘he healty eferonco intra! of gluccse. 4. Compare fype 1 and type 2 clabetes melts with regard to rovalence, cause, ag at onset, symptoms, and laratory vas, 5. Slate the basi ciara forthe dagnoss of ciabetas metus, including American Diabetes Association guidelines. 6, Discuss the abnormal metabolic relationships among glucose, Ketones faty acids, and metalic acids in an insuln-deficent individual, 7, Outine the procedure for administration ofan oral glucose tolerance test and interpret tho resus. 8, Ust the laboratory procedures inolved in assessment of dabetes| rmefitus in a nonpregnant indica 9, Ust three causes of hypogyceria. 10, List four methods of serum glucose anaiysis, sate the specimen requirements and principles ofeach, and list the kaown interferences In cach, 11 Define clycated hemogicin and hemogabin A, state the clinical uty of ts measurement, and Ist ree methods of ayeated hemogibin analysis. 12, Resolve caso studs rogarcing carbohyarate analy n the ‘assessment of carbonydrate concer, KEY WORDS AND DEFINITIONS ‘Advanced Glycation End Praluets (AGE): Proteins that hhave been irreversibly modified by nonenaymatic attachment of glucose; may contribute to the chronic complications of diabetes Carbohydrates: Neutral compounds composed of catbon, hydrogen, and oxygen (in a ratio of 1:2:1) that constitute a major food clas Diabetes Mellitus: A group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized, producing hyzerglycemia. Carbohydrates David B. Sacks, M.B., Ch.B, F.R.C.Path. Diahetoyenes: Genes that contribute to the development of diabetes a genetic basis is identified in fewer than 5% of individuals with tye 2 diabetes Gestational Diabetes Mellitus (GDM): Carbohydrate intolerance that arses during pregnancy. Glucose: A six-earbon simple sugar that isthe premier fuel for most organisms and an important precursor of other body constituents. Glycated Hemoglobin: Hemoglobin that has a sugar residue attached Hb A. is the major fraction (~80%) of glyeated hemoglobin; also known as gycohemoglabin Glycogen: A polysaccharide having a formula of (CHigOs)n, used by muscle and liver for carbohydrate storage Hyperglycemia: Increased glucose concentrations in the blood. Hypoglycemia: Decreased glucose concentrations in the blood. Insulin: A protein hormone produced by the Becells of the pancreas thar decreases blood glucase concentrations. Ketones: Compounds that arise from free fatty acid breakdown; insulin deficiency leads to incressed serum, ketones, which are the major contributors o the metabolic acidosis chat occurs in individuals with diabetic ketoacidosis, Lactate: An intermediary product in carbohydrate metabolism that accumulates in the blood predominantly when tissue oxygenation is decreased; an increased blood lnctate concentration is called lactic acidemia, and may be associated with lactic acidesis. Microalbuminuria: A rate of excretion of alburin in the urine (20 to 200 jglmin) that is heeween normal andl overt proteinuria; increased urinary excretion of albumin precedes and is highly predictive of diabetic nephropathy. lstributed in plants and animals. They perform mul ‘tile functions, such as being structural components as in RNA and DNA (cibose and denxyribose sugats) and provid. ing a source of energy (glucose). Glucose is ceived fram (1) the breakdown of carbohydrates in the diet (grains, starchy vegetables, and legumes) or in body stores (glycogen), and (2) endogenous synthesis ftom protein or from the glycerol moiety of triglycerides. When energy intake exceeds expendi- ture, the excess is converted to fat and glycogen for storage in adipose tissue and liver or muscle, respectively. When energy expenditure exceeds calorie intake, endogenous glucose forma: tion occurs from the breakdown of catbohydmte stores and from noncarbohydrate sources (e.g, amino acid, lactate, and alycerol). Ce including sugar and starch, are widely 313318 PART IV. Analytes Insulin, glucagon, and epinephrine maintain the glucose concentration in the blood within a fairly narrow interval under diverse conditions (feeding, fasting, of severe exercise) Measurement of glucose is one of the most commonly pet- formed procedures in hospital and other healthcare chemistry laboratories, The most frequently encountered disorder of car- bbohydrate metabolism is high blood glucose due to diabetes ‘mellitus, which affects approximately 8% of the U.S. populae tion, The incidence of hypoglycemia (low blood glucose) is unknown, but is substantially lower. cH ssisisiaaa ‘Carbolyydrates are aldehyde or ketone derivatives of polyhy- roxy (more than one —OH group) alcohols, or compounds that yield these derivatives on hydrolysis. Monosaccharides ‘A monosaccharide i a simpfe sugar, which consists ofa single polyhydroxy aldehyde or ketoneunitand is unable to be hydro- lyzed to a simpler form. The beckbone is made up of several ‘carbon atoms. Sugars containing three, four, five, six, and seven carbon. atoms are known as trios, teroses, pentoses, hexoses, and heptnses, respectively. One of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group. ‘An aldehyde has the carbonyl group at the end of the carbon ‘chain, whereas if che carbonyl group is at any other position, aaketone is formed (Figure 22-1). The simplest carbohydrate is glycol aldehyce, the aldehyde derivative of edhylene glycol. ‘The aldehyde and ketone derivatives of glycerol are, respec tively, glyceraldehyde and dihyckoxyacetone (see Figure 22-1), Monosaccharides that are aldehydes or Ketones are called, respectively, aldoses and ketoses (Figure 22-2). Compounds that are identical in composition and differ only in spatial configuration are called stereoisomers. The carbon ators in the unbranched chain are numbered I to 6, as shown by the numbersat the tof the formula for D-glucase in Figure 22-2, The designation D- or 1- refers to the position of the hydroxyl group on the carbon atom adjacent to the last (bottom) CH.OH group. In general, the designation of n- and I> for a sugar molecule refers tothe stereoisomerie forms of the “=o peo Pace now bon buon Dost Caycotatehyde — Glyewalbiyde —Diyerayaeane ‘osa) ‘acoes) Figure 22-1 Two- und thee-carhon carbohytrates, nen0 emo cHoH Ho=0 2 mbon wodu bo tow sword ton nota wou 6 mdion wo-br ation nob s ndion not wton ton «© bxon don axon bao i Figure 22-2 Typical siccatbon sugars, hhighest-numbered asymmetrical carbon atom.* Br convention the D-sugars are written with the hydroxyl group on the right, and the Lesugats are written with the hydroxyl group on the lefe (see Figure 22-2), Most sugars in the humar body are of the v-configuration. A number of different structures exist, depending on the relative positions of the hydroxyl groups on the carbon atoms. The formula for glucose can be written in the form of either aldehyde or enol, a shorelived reactive species. Shift to the enol anion is favored in alkaline solution, 2s follows 0 gon E08 ution — fon teat Lon yao. Ae al Basle ‘The presence ofa double bond and a negative charge in the ‘enol anion makes glucose an active reducing substance that is oxidized by velatively aild oxidising agents sich as cupric (Car*) and ferric (Fe") ions. Glucose in hot allaline solution readily reduces cupric ions to cuprous ions. The color change has been used as a presumptive indication for the presence of slucase, and for many years, blood and urine glucose were ‘measured this way. Many other sugars also reduce cupric ions in alkaline solution, and these are collectively zferred to as reducing sugars ‘The aldehyde group reacts with the hydroxyl group on carbon 5, represented by a symmetrical ring structure and depicted by the Haworth formula, in which glicose is consi cred as having the same basie structure as pyran (Figure 22-3), In this formula, the plane of the ving is considered to be per- pendicular to the plane of the paper, with the heavy lines pointing toward the reader. Hydroxyl groups in rosition 1 are then below the plane (o-canfguration) or above the plane (Beconfiuration). A six-member ring sugar, coataining five ‘carbons and one oxygen, isa derivative of pyran and is called f pyramose, When linkage occurs with formation of a five- member ring, containing four carbons and one oxygen, the sugar has the same basic structure as furan and is alled a fra- nose. Fructose is shown in two cyclical forms. Frictopyranose 4s the configuration ofthe fre sugar, and fructofuranose occurs whenever fructose exists in combination with disaccharides and polysaccharides, as in sucrose and inulin, Disaccharides ‘Two monosaccharides join covalently by an O-elycosidic bond, with the loss of a molecule of water, to form a disaccharide, “The chemical bond between the sugars always involves the aldehyde or ketone group of one monosaccharide joined to analcohol group (e.g, maltose} or an aldehyde orketone group (4, sucrose) of the other monosaccharide (Figure 22-4). The ‘most common disaccharides are as follows Maltose = glucose + glucose Lactose = glucose + galactose Sucrose = glucose + fructose *Although the 0 and 1 designations are used in this chapter, sealers should be aware that in the Cahn-lngold-Prelog system a series of rules determines configurations. In this new system the symbols R and S are used to designate configarations,Carbohydrates, CHAPTER 22 378 1,08 0-Ghcopyranose Pyros Pym since H,081 ou ep Frcopyrnose “e Gt on 108 me Sen Ss “i yen Ral See ea on A sire e-p-Frstne (ep ucirmone Figure 22-8 The Havorth formula for sugars. 108 081 ul Hon ¥ f ou on) Con > : one a HOH Rabing e-o-Maow = (.0-0-Gluopyrnosy (1-0 gucopytanse rt eon oe on a one” ben > LRe pe in tbe potas (.f-0-Galatopyansy(1-r0-8--sheopyranose eH08 ou on scose O-xo-Gtucopyenagyi{-)o-taconeanse Figure 22-4 Structural formulas of disaccharides If the linkage between two monosaccharides & beeween the aldehyde or ketone group of one molecule ancl a hydroxyl group of another molecule (as in maltose and lactose), one Potentially free kerone or aldehyde group remains on the second monosaccharide. Consequently, the second glucose residue can be oxidized and is capable of existing in a- or B- pyranose forms. Thus the disaccharide isa reducing sugar, but its reducing power is only approximately 40% of the reducing power of the two single monosaccharides addled cogether. Altematively, if the linleage between two monosaccharides involves the aldehyde or ketone groups of both molecules (as {in sucrose), a nonreducing sugor results because no free alde- hyde of ketone group remains, Polysaccharides ‘The linkage of multiple monosaccharide units results in the formation of polysaccharides. The major storage carbohydrates are starch int plants and glycogen in animals, both of which form granules inside cells. Polysaccharides can provide struc tural support. Cellulose is used hy plants, whereas chitin is the principal component of the exoskeleton of arthropods (insects and crustacea). Starch and Glycogen Most starches are composed of mixture of amyloses and amylopectins. Amylose consists of one long unbranched chain ‘of glucose units linked together by ct-1,4-linkages, with only the terminal aldehyde group free, In amylopectin, most of the units are joined by o-1,4-inks, but 0-1,6-slycosidic bonds also exist every 24 to 30 residues, producing side chains. Amylopectin contains up to L million glucose residues. The structure of glycogen is similar to that of amslopectin, but branching is more extensive and occurs every 8 to 12 glucose residues. These branches enhance the solubility of glycogen and allow the glucose residues to be more readily mobilized, Glycogen is most abundant in the liver and ako is found in skeletal muscle. The difference in structure between amylose and amylopectin is important in selection of the appropriate starch substrate for amylase determinations (see Chapter 19), ‘The rate of hydrolysis is affected by steuctural diferences in the starch, Cellulose Cellulose is an important structural polysaccharide in plants, Ie is an unbranched polymer of glucose residues joined by B- 1 4-linkages. The B-configuration facilitates the formation of long straight chains, producing fers of high tensile strength. ‘The B-l,4-linkages ate not hydrolyzed by ceamylases. Because Thumans'do not have cellulases, they are uneble to digest ‘vegetable fiber. Glycoproteins ‘Many integral membrane proteins have oligosaccharides cova- lently attached to the extracellular region, forming glycopro- teins. In addition, most proteins that are secieted, such as ‘antibodies, hormones, and coagulation factors, are glycopro- teins, The number of attached carbohydrate residues varies among proteins andl constitutes 19% t0 70% of the weight of the glycoprocein. The oligosaccharides are attached by O- slycosidic linkages to the side chain oxygen of serine or threo- nine tesidues or by N-glycosidic linkages to the side chain nitrogen of asparagine residues.376 PART IV Analytes One biotogical function of the carbohydrate chains is to regulate the lifespan of proteins. For example, loss of sialic acie residues from the end of oligosaccharide chains on erythrocytes resus in the removal of red blood cells from the circulation. Carbohydrates also have heen implicated in cell-cell recogni- tion, in secretion, and in targeting of proteins to specific sub- cellular domains. Glycoproteins of special interest are glycated hemoglobin (GHb) and similar proteins, which are used to monitor long- term ghucose control in people with diabetes mellitus. In,addi- tion, GHb is a measure of the risk for the development of complications of diabetes Chemically, glycation is the nonenzymatic addition of a sugar residue tb amino groups of proteins, Human adult hemo- slobin (Hb) usually consists of Hb A. (97% of the total), Hb A; (2.5%), and Hb F (0.5%). Hb A is made up of four poly- peptide chains, two a and two Bechains. Chromatographic snalysis of Hb A identifies several minor hemoglobins, namely Hb A,., Hb An, and Fb A,,, which collectively are referred toas Hb A,, faschemoglobins (because they migrate more rapidly than Hb Ain an electrical fed), glycohemoylobins, or glycated hemoglobins. The Joint Commission on Biochemical Nomen- clature ofthe Incernationall Uninn of Pure and Applied Chem- istry recommends the term neodycoprotein for such derivatives and the term ghcatin to describe this process. Therefore although glycosylated and glucosyated have been widely used in the literature, the term gycated is preferred. Hb Axcis formed by the condensation of glucose with the N-terminal valine residue of either B-chain of Hb A to form an unstable Schiff base (aldimine, pre-Elb Ay. Figure 22-5), The Schiff base may either dissociate or undergo an Amadori rearrangement t© form a stable ketoamine, Hb Aj. Hb Ata and Hb Asis which take up Hb A, have fructose-l 6-diphosphate and glucose- G-phosphate, respectively, attached to the amino terminal of the chain. Hb An, identified by mass spectrometry, contains pyruvic acid linked to che amino terminal valine of the B- chain, probably by a ketamine o enamine bond. Hb Ay. isthe ingjor fraction, constituting approximately 80% of Hb Ay Glycation may also occur at other sites on the B-chain, such as on lysine residues, and it may occur on the a-chain, These GHbs cannot be separated from nonglycated hemoglobin by methods based on charge (suchas ion-exchange chromatogra- phy}, but are measured by boronate affinity chromatography. CHEM! ANC ee ‘Glucose isthe primary energy surce for the human body. After absorption (see Chapter 37), the metabolism of all hexoses ‘proceeds according to the body's requirements, This metabo- lism results in (1) energy production by conversion to carbon dioxide and water, (2) storage as glycogen in the liver or rr Gc eek) Ketamine ui emo wc ga mb H.CNIBA he don won BOA + Geom 42 hours), sluconeogenesis accounts for essentially all the glucose produc- tion. In contrast, after a meal the absorbed glucose is converted to glycogen (for storage in the liver and skeletal muscle) or fat {for storage in adipose tissue), Despite large fluctuations in the supply and demand of car- bohydrates, the concentration of glucose in the blood is nor- rally maintained within a narrow interval by hormones that modulate the movement of glacose within the body. These include insulin, which decreases blood glucose, and the coun- terregulatory hormones (glucagon, epinephrine, cortisol, and growth hormone), which increase blood glucose concentra- tions (Figure 22-6). Normal glucose disposal depends on (1) the ability ofthe pancreas to secrete insulin, (2) the ability of insulin t© promote uptake of glucexe into peripheral tissues, and (3) the ability of insulin to suppress hepatic glucose pro- duction. The major insulin tanget organs are the liver, skeletal mascle, and adipose tissue. These organs exhibit some differ fences in theit responses to insulin. For example, insulin stra Tates glucose uptake through a specific glucose transporter, GLUT4, in muscle and fat cells but not liver cell Regulation by Insulin of Blood Glucose Concentration Insulin is a protein produced by the B-cells of the isles of Langerhans in the pancreas, Insulin was (1) the frst proteinCarbohydrates. CHAPTER 22 a1 Somatostatin ~ Growth Rormone Caco i Tipoganesis [Giucose urake Figure 22-6 Hormonal regulation of .Giveose up| ‘atyeoysts. blood glucose. Cortisol growth hormone, and epinephrine also antagonie the effect of inalin. +, Stimulation; ~ inhibition. Liver ‘Adipose tissue Buscie hhormone to be sequenced, (2) the fist substance t0 be mene sured by radioimmunoassay (RIA), and (3) the first compound produced by recombinant DNA technology for practical ws. Iisan anabolic hormone that stimolates the uptake of glucose into fat and muscle, promotes the conversion of glucose to alycogen or fat for storage, inhibits glucose production by the liver, stimulates protein synthesis, and inhibits protein break- down, The release and mechanism of action of insulin are more fully discussed on pages 843-848 in an expanded version ofthis chapter.” Human insulin (molecular mass $808 Da) consists of 5 ‘amino acids in two chains (A and B) joined by two disulfide bridges, witha third disulfide bridge within the A chain. Insulin from most animals is similar immunologically and biologically to human insulin, and in the past patients were treated with insulin purified from beef or pig pancreas. Virtuslly all patients are now treated with recombinant human insulin, Preproinsulin, a protein of about 100 amino acids, is noe detectable in the circulation under normal conditions because it is enzymatically cleaved and converted to proinsulin, Proin- sulin is stored in secretory granules in the Golgi complex of the B-cells, where proteolytie cleavage to insulin and connect- ing peptide (C-peptide) occurs This posttranslational process- ing is catalyzed by two Ca"-regulated endopeptidases, namely prohormone convertases 1 and Z (PCI and PC2), The splic proinsulin intermediates, split 32:33 proinsulin and split 65,66 proinsulin, are further hydrolyzed to insulin and C-peptide. At the cell membrane, the insulin and C-peptide are released into the portal circulation in equimolar amounts, In addition, small amounts of proinsulin and intermediate cleavage forms enter the circulation, Proinsulin, which has relatively low biological activity {approximately 10% of insult potency), is the major storage form of insulin, Normally, only siall amounts (about 3% of the amount of insulin, on a molar basis) of proinsulin enter the circulation. Largely because the hepatic clearance of proinsulin is only 25% of insulin clearance, the halE-life of proinsulin ‘is twofold to threefold loager, and concentrations in the fasting stare are approximately 10% to 15% of insulin C-peptide isbelieved to have biological activity, and appears necessary to ensure the correct structure of insulin. Although insulin and C-peptide are secreted into the portal circulation {n equimolar amounts, fasting concentrations of C-peptide are fivefold to tenfold higher than these of insulin due 1 the longer half-life of C-peptide (about 35 minutes). The liver does not extract C-peptide, which is removed from the circulation by the kidneys and degraded, with a fraccion excreted unchanged in the urine Glucose Transport (One of the fndamental effects of insulin is to increase glucose uptake into cells. The molecular mechanism of insulin action is extremely complex. The transport of ghicose into cell is ‘modulated by two families of proteins. The intestinal sodium aandjor glucose cotransporter promotes the uptake of glucose and galactose from the lumen of the small bowel and their reabsorption fronn the urine in the kidney. The second! family ‘of, glucose carriers, termed faciltarve ghicore mansporters (GLUTS), is located on the surface ofall cells (Table 22-1), ‘These transporters are designated GLUTI to GLUTIZ, bused con the order in which they were identified. On the Basis of sequence similarities, chey can be divided into three subfami- lies, namely class 1 (GLUT 4), clas {I (GLUT 5, 7, 9, and 11), and clas III (the newly described GLUT 6, 3, 10, and 12). GLUT! is widely expressed and provides many cells with theit bosal glucose requirement. GLUT in the blood-beain barrier and GLUT3 in neuronal cells provide the constant high con- centrations of glucose required by the brain, GLUT2 is ‘expressed in hepatocytes, B-cells of the pancreas, and basolat- eral membranes of intestinal and renal epithelisl cells. It is @ low-afinity, high-capacity transport system that allows non= rate-limiting, movement of glucose into and trem these cell GLUI4 catalyzes the eate-limiting step for glucose uptake and metabolism in skeletal muscle, the major ongan of glucose consumption. When circulating insulin concentrations are378 PART IV Analytes Bees a co Namo ‘Tissue g Funston GLUT (enthrocye) ‘Wide distribution, especialy bran, Kdoy, clon, and fla ssies Basal ucose transport LUTE ven) Liver, B-calls of pancreas, smal intestine, and Kidney Non-rate-imitng gucose transpot GLUTS fran) ‘ide dstibuton, especialy neurons, placenta, and testes ‘Guecse anspor in neurons LUTE (muscle) Shell muscle, cardlac muscle, and adipose tissue Insulnstinuated glucose anepart GLUTS (smal intestine) Small ntestine, Keys, skoelal muscle, brain, and adlpose tissue Fructose tran (not glucose) ours Leukooyts and bain Glucose aur Liver Release of glucose from endoplasmic reticulum curs Testes blasloosis, bran, muscle, and adipose tissue Gluence transport Sura Liver and kidnoys curio vec and panctees suri cart and sklotal muscle Glucose transport auuri2 Shell muscle, eardac muscle, adipose tissue, and breasts low, most of the GLUT is localized in intracellular compart- ments and is inactive. After a meal the pancreas releases insulin, which stimulates the translocation of GLUTA to the plasma membrane, thereby promoting glucose uptake into slel- etal muscle and fat. Insulin-stinulated glucose transport into skeletal muscle is impaired in irdividuals with type 2 diabetes mellitus, but_the mechanism of the defect has not been established. GLUTS is esponsible for fructose uptake in the intestine. Less is known about the other GLUTs. Glucose transport has been reported for GLUT 6, 8, II, and 12. Formation of Glycated Hemoglobin Formation of GHb is essentially irreversible, and the concen- tration in the blood depends on both the lifespan of the red blood cell (average 120 days) and che blood glucose concentra- tion. Because the rate of formation of Hb is directly propor- tional to the concentration of glucose in the blood, the GHb camcontration represents the integrated values for glucose over che preceding 6 0 8 weeks. This provides an additional criterion for assessing glucose contol because GHb values are free of day- to-day glucose fluctuations and are unaffected by recent exet- cise or food ingestion. The contribution of the plasma glucose concentration ata given time point to the ultimately measured Gib depends on the time interval before blood is sampled the concentrations of glucose at recent time points provide a larger contribution to GHb than do earlier values, at least partly because more of the red cells survive ftom the recent time point than from che mote remote time point. The plasma glucose in the preceding | month determines 50% of the Hb Ai, whereas days 60 to 120 determine only 25%. After a sudden alteration in blood glucose concentrations, the rate of change of Hb Aig israpid during the initial 2 months, followed by a more gradual change approaching steady state 3 months later. The half-time is 35 days. ‘The interpretation of GHb depends on the red blood cells hhaving a normal lifespan. Patients with hemolytic disease or other conditions with shortened red blood cell survival exhibit a substantial reduction in GH.’ GHb concentrations ean still be used to monitor these patients when their red cell survival is not changing, but values must be compared with previous values from the same patient, not published reference inter- vals, Individuals with recent sigificant blood loss have falsely low values owing to a higher fraction of young erythrocytes; GHb will then increase asthe cells age, allowing time for the hemoglobin to have glucose attached to form GHb. High GHb concentrations have been reported in iron deficiency anemia, probably because of the high proportion of old erythrocytes ‘The effect of hemoglobin variants (such as Hb F, Hb S, and Hb C) depends on the specific method of analysis (discussed later)? Depending on the particular hemoglobinopathy and assay method, results may be spurious increased or decreased. ‘Another source of error in selected methods is carbamoylated hemoglobin. This is formed by attachment of urea end is present in Targe amounts in renal failure, which is comwaen in patients with diabetes. Counterregulatory Hormones Several hormones have actions opposite those of insulin, These counterregulatory hormones are catabolic and increase hepatic sucose production initially by enhancing the breakcown of alycogen to glucose (glycogenolysis) and ler by stimulating the synthesis of glucose (gluconeogenesis). The body's initial response (within minutes) to low blood glucose isan increase in glucose production, stimulated by glucagon and epineph. sine. With time (3 to 4 hours), growth hormone and cortisol increase glucose mobilization and decrease glucose use (sce Figure 22-6), Evidence also suguests that glucose production by the liver is an inverse function of ambient ghicose concentra tion, independent of hormonal factors (glucose autoregula- tion}, The role of other hormones or neurotransmitters is not cleas, but appears relatively unimportant. The mahtiple coun- rerregulatory hormones exhibit both redundancy ancl hierar chy. Glucagon i the most important, and epinephrine becomes critical when glucagon is deficient, ‘The other factors have lesser roles Glucagon Glucagon is @ 29-amino-acid polypepride secreted by the 0: cells of the pancreas. ‘The major targec organ fo: glucagon is the liver, where it binds to specific receptors and increases intracellularadenosines’-monophosphate (AMP)and calcium. Glucagon stimulates the production of glucose ir. the liver by alycogenolysis and gluconeogenesis (see Fiyure 2-6). In add tion, glucagon enhances Ketogenesis in the liver. A minor target organ for glucagon is adipose tissue, where -he hormone increases lipolysis. Glucagon secretion is primarily regulated by plasma glucose concentrations, low and high plasma glucose concentrations being stimulatory and inhibitory, respectively. Long-standing diabetes mellitus results in an impaired gluca- gon response to hypoglycemia, increasing the incidence ofCarbohydrates, CHAPTER 22 379 hypoglycemic episodes. Stress, exercise, and amino acids also induce glucagon release. Insulin inbibits glucagon release from. the pancreas and decreases glucagon gene expression, thereby decreasing its biosynthesis, Increased glucagon concentrations, secondary to insulin deficiency, are thought to contuibute to the hyperglycemia and ketosis of diabetes. Epinephrine Epinephrine (adrenaline), a catecholamine secreted by the accenal medulla, stimulates glycogen breakdown (glycogenoly- sis) and decreases glucose use, thereby increasing blood glucose concentrations. It also stimulates glucagon secretion and inhibits insulin secretion by the pancreas (see Figure 22-6), thus further increasing blood glucose, Epinephrine appears to play a key role in glucase cointerregulation when glucagon secretion is impaired (e.g, in cases of type I diabetes mellitus), Physical or emotional stress increases epinephrine production, celeasing glucose for energy. Tumors of the adrenal medulla, known as pheochromocytomas, secrete excess epinephrine of rorepinephrine and produce moderate hyperglycemia as long as glycogen stores are available in the liver Growth Hormone Growth hormone is a polypeptide secreted by the anterior pituitary gland (see Chapter 40). It stimulates giconeogenesis, enhances lipolysis, and antagenises insulin-stimulated glucose uptake. Cortisol Cortisol, secreted by the adrenal cortex in response to adreno- corticotopic hormone (ACTH), stimulates gluconeogenesis and increases the breakdown of protein and fat (see Chapter 39). Hyperplasia or tumors of the adrenal cortex ean proxluce cortisol and increase its concentration (Cushing syadrome), thus leading to hyperglycemia, In contrast, those with Addison disease demonstrate adrenocortical insufficiency because of destruction or atrophy of the adrenal cortex and may exhibit hypoglycemia. Other Hormones Influencing Glucose Metabolism ‘Thyroxine and somatostatin so affect glucose metabolism. Thyroxine ‘Thyroxine, secreted by the thyroid gland, is not directly involved in glucose homeostasis but stimulates glycogenolysis and increases the rate of gastric emprying and intestinal glucose absorption (see Chapter 41). These factors may produce glucose intolerance in chyrotoxic individuals, but such a person usually has a normal fasting plasma glucose concentration. ‘Somatostatin ‘Somatostatin, also called growth hormone-inhibiting hormone ‘and, historically, somatotrophin release-inhibiting. factor (SRIF), isa [4-amino-acid peptide found in the gastrointesti- nal tract, hypothalaraus, and the 5-cells of the pancreatic islets. ‘Although somatostatin does not appear to have a direct effect ‘on. carbohydrate metabolism it inhibits release of growth hormone from the pituitary. In addition, somatostatin inhibits secretion of glucagon and -nsulin by the pancreas, thus modulating the reciprocal relationship hetween these two hormones. Ketone Bodies ‘The development of ketosis requires changes in both adipose tissue and the liver, The primary substrates for ketone body formation are free fatty acid from adipose stoes. Normally, long-chain fatty acids are taken up by the liver, reesterfied to triglycerides, and stored in the liver or converted to very- low-density lipoproteins and returned to. the plasma. In individuals with uncontrolled diabetes, che low insulin con ‘centrations result in ineeeased lipolysis ancl decreased reesteri- fication of fatty acids into triglycerides, thereby increasing plasma free fatty acids. Moreover, the patient's increased glu- cagon-to-insulin ratio enhances fatty acid oxidation in the liver. Thus increased hepatic ketone proxluction and decreased peripheral tissue metabolism lead to acetoacetate accumula- tion in the blood. A small fraction undergoes spontaneous decarboxylation to form acetone, but the majority is converted to B-hydroxybutyrate: re frogs Oa 0? stpsepne ne ba, ST di of en, mtr Lae nop ‘esos peas & Stee a co I ae ‘The relative proportions in which the three ketone bodies are present in blood vary, depending on the redox state of the cell In healthy individuals, B-hydroxyburyrate and acetoace- tate, which are present at approximately equimolar concentra tions, constitute vieeully all che serum ketones. Acetone is 2 ‘minor component. In cases of severe diabetes, the ratio of B- hhydroxybutyrateto acetoacetate may increase upto6: I because of the presence of lange amounts of the reduced form of nicotinamide adenine dinucleotide (NADH), which favors B- hydroxybutyrate production. Lactate and Pyruvate Lactic acid, an intermediary in carbohydrate metabolism, is predominantly derived from white skeletal muscle, brain, skin, renal medulla, and erythrocytes. The blood lactate coneentea- tion depends on the rate of production in these tissues and the tate of metabolism in the liver and kidneys. The liver uses approximately 65% (75 giday) of the total basal lactate pro- dluced predominantly in gluconeogenesis. The Cori cycle isthe conversion of glucose to lactate in the periphery and reconver sion of lactate to glucose in the liver. Extrahepatic removal of lactate is by oxidation in red skeletal muscle and the renal cortex. A moderate increase in lactate production results in increased hepatic lactate clearance, but uptake by the liver is saturable when concentrations exceed 2 mmol/L. During stren: ‘uous exercise, for example, lactate concentrations may increase significantly, from an average concentration of about 0.9 to ‘more than 20 mmol/L, within 10 seconds, No concentration of lactate is uniformly necepted for the diagnoss of lactic aci- dlsis, but lactate concentrations exceeding 5 mmol/L and with DH less than 7.25 indicate significant lactic acidosis.380 PART IV. Analytes CLINICAL SIGNIFICANCE jabetes mellitus and hypoglycemia are clinical conditions asoviated wth abnormal eabhyrare metabelise, Diabetes Mel Diabetes mellitus is a group of metabolic disorders of carbohy- date metabolism in which glucose is underutilized, producing hhyperalycemia. Some individuals may experience acute life- threatening hyperglycemic epics, such. as ketoacidosis ot hyperosmolar coma, As the disease progresses, individuals are at increased risk for the development of specific complications, luding retinopathy (which may lead to blindness), renal failure, neuropathy (nerve damage), and atherosclerosis. The {ast condition may result in stroke, gangrene, or coronary artery disease In 1987 the prevalence of dingnosed diabetes was 6.8 million. In 2001 the U.S. Centers for Disease Control and Prevention (CDC) estimated a prevalence of 7.9% in adults oc 16.7 million people. Because at leat 30% ofall prevalent cases ate undiagnosed, i¢ was thought that the total nuaber may have been almost 22 millicn. This increase in the preva- lence of diahetes isa global phenomenon as the prevalence of diabetes in adults worldwide was estimated to be 4.0% in. 1995 and is anticipated to tse to 5.4% (300 million adults) by the year 2025. OF the 300 million adults, yreater chan 75% will live in developing countries. ‘These statistics have led to dia- betes heing described as “one of the main threats to human health in the cwenty-first century.” The prevalence of diabetes mellitus ineseases with age, and approximately half of all cases ‘occur in people older than 55 years. In the United States, ~20% of the population older chan 65 years have diabetes, ‘There is racial preditection, and by the age of 65, 33% of His- panics, 25% of blacks, and 12% af whites in the United States will have diabetes mellitus. In 2002 diabetes mellitus was esti- tated to be responsible for $132 billion in healthcare expen: ditures in the United States, The direct costs were $92 billion, with 50% of that incurred by trose older than 65 years. An «estimated 186,000 deaths annually are attributable to diabetes with American women twice zs likely to die from diabetes ‘nelitus as from breast cancer. Approximately one in five ‘American healtheare dollars spext in 2002 was for people with diabetes mellitus Classification Diabetes was initially diagnosed by the oral glucose tolerance test (OGTT). In 1979 a work group of the National Diabetes Data Group proposed modified criteria for diagnosis. This clas- sification scheme recognized twomajor forms of diabetes—type [ (insulin-dependent) diabetes nellius (IDDM) and type IL (con-insulin-dependent) diabe:es mellitus (NIDDM). The terms juverle-onse diabetes andl adult-onset diabetes were abol- ished. To hase the classification on etiology rather than rreat- rent, in 1995 the American Diabetes Association (ADA) established a work group to reexamine the classification and diagnosis of diabetes mellirus. The revised classification, pub- lished in 1997, eliminates the tsrms insulindependent diabetes rellius and non-insulin-dependers diabetes melicus, which now are termed type I diabetes and tyfe 2 diabetes, respectively (Box 22-1). Another siguificant change is de elimination Uf dhe categories of previous abnormality of glucose tolerance and potential abnormality of glucose tolerance. 80x 22-1 | Classification of Diabotes Mellitus and Other Categories of Glucose Intolerance ‘Type + diabetes ‘A immune mediated B. ldopatic Type 2 abetes Other spectic types of dabetes ‘Getaonal dabetes matiis (60M) limpaied lucas tolerance (GT) limpared fasting glucose (FS) From te American Diabetes Assoditon. Retort of the exper cormsise on he digi and clasifcion of tes metas, Drs Cre 1997/20, 1183-1201 Type 1 Diabetes Metitus ‘Type 1 diabetes mellitus was formerly known as IDDM, type I, or juvenile-onset diabetes. Approximately 5% to 10% of all individuals with diabetes mellitus are in this category. Symp- toms (eg. polyuria, polydipsia, and rapid weight loss) usually present acutely; individuals have insulinopenia {adeficiency of insulin) because of loss of pancreatic islet B-cells and depend ‘on insulin treatment to sustain life and prevent ketosis. Most individuals have antibodies that identify an autoimmune process (see later discussion); some have no evidence of auto- immunity and are classified as tybe 1 idiopathic. The peak incidence of this disease is in childhood and adolescence ‘Approximately 75% acquise the disease hefore 30 years of age, but the onset in the remaining percentage of individuals may feccur at any age. Age at presentation is not @ criterion for clasiication, Type 2 Diabetes Metlitus Formerly known as NIDDM, type 2 diabetes consticutes approximately 90% of all cases of diabeses. Patiats (1) have minimal symptoms, (2) are not prone to ketosis, and (3) are ‘not dependent on insulin to prevent ketonuria, Ins concentra tions may be within the reference inter, decreased, o creased and most people with this form of diabetes have impaired insulin action. Obesity is commonly associated, and weight loss alone usually improves the hyperglycemia. However, many individuals with type 2 diabetes may require dieta-y manipula- tion, an oral hypoglycemic agent, or insulin therapy to control byperalycemia. Most patients acquire the discase after age 40, butitmay occur in younger people. Type 2 diabetes in children and adolescents is an emerging, significant problem. Among children in Japan, type 2 diabetes is naw more common than ‘ype | Gestational Diabetes Melitus Gestational Diabetes Mellitus (GDM) is carbohydrate intol- ceximce of variable severity wih onset or fst reopitondrng pregency.” Note that wornen with diabetes who Fecame preg- nant are not included in this eategory. Estimates of the fre- quency of abnormal glucose tolerance during pregnancy range from 1% to 14%, depending on the population studied and the agnostic rests employed, In the United States, GDM occurs in 6% to 8% of pregnancies. Women with GDM are at a sig- nificantly increased risk of subsequent diabetes, predominantlyCarbohydrates. CHAPTER 22 3B1 type 2. The cumulative incidence of type 2 diahetes after GDM varies among populations, anging from ~40% to 70%. The annual incidence is markedly increased above that in the general population and rises during the fist 5 years, reaching a plateau after 10 years. At 6 to 12. weeks posipartum, all patients who had GDM shoul be evaluated for diabetes and, if diabetes is aor present, be reevaluated for diaberes at least every 3 years Other Specific Types of Diabetes Metitus ‘This subclass includes patients in whom hyperglycemia is due toa specific underlying disorder, such as (1) genetic defects of B-cell function; (2) genetic defects in insulin action; (3) disease ‘of the exocrine pancreas; (4) endocrinopathies (e.g, Cushing disease, acromegaly, and glacagonoma); (5) the administration ‘of hormones of drugs known to induce Prcell dysfunction (eg, dilantin and pentamidine) or impair insulin action (eg. ‘glucocorticoids, thiazides, anc Bradrenergics); (6) infections; (7) uncommon forms of immune-mediated diabetes; or (8) other genetic conditions (e4, Down syndrome, Klinefelter syndrome, and porphyria; see ADA" for a detailed list). This was formerly termed secondary diabetes. Impaired Glucose Tolerano> Ipaired glucose tolerance (IGT) is diagnosed in people who have fasting blood ghicose concentrations less than thase required fora diagnosis of diabetes mellitus, but have a plasma glucose response curing the OGTT between oral and dia- betic states. An OGTT is required to assign a patient co this class. Development of overt dabetes occurs ata rate of 1% to '5% per year in people with IGT, but a large proportion spon- taneously revert to normal glucose tolerance, Microvascular disease is quite tare in this group, and patients usually do not experience the renal or retinal complications of diabetes Patients have an increased prevalence of atherosclerosis ond mortality from cardiovascular disease. Impaired Fasting Glucose Impaired fasting glucose (IRG) is analogous to IGT, but is diagnosed by a fasting glucose value above normal but below the concentration for diagnosis of diabetes. [tis @ metabolic stage between normal glacose homeostasis and diabetes. As with IGT, persons with IFG are at increased risk for the devel- ‘opment of diabetes and cardiovascular disease. IFG and IGT ‘are not clinical entities, but rather risk factors for diabetes and cardiovascular disease. Pathogenesis of Type 1 Diabetes Mellitus Mose type 1 diabetes mellitus results from a cellolar-mediated autoimmune destruction of the insulin-secreting cells of pan- creatic B-cells In most patients, the destruction is mediated by T cells, Ths is termed type LA or immune-mediated diabe- tes, The o-, 8, and other islet cells are preserved. The islet cells have a chronic mononuclear cel infiltrate, called insu ‘The autoimmune process leading to type 1 diabetes begins months or years before the clinical presentation, and an 80% to 90% reduction in the volume of the Breels is required to induce symptomatic type 1 ciabetes. The rate of islet cell destruction is variable and is usually more rapid in children than in adults, Antibodies Circulating antibodies are markers of B-cell autoimmunity, which are detected in the serum years before the onset of hyperelycemia. They include" 1. Islet cell cytoplasmic antibodies (ICAs) react with a sialoglyeoconjugate antigen present i the cytoplast of ail endocrine cells of the pancreatic islets. These antibodies are detected in the serum of 0.5% of normal subjects and 75% to 85% of patients with newly diagnosed type 1 diabetes. 2. Insulin auzoanaibodios (LAAs) are present in more than 90% of children who develop type I diabetes before age 5, but in fewer than 40% of individuals developing, diabetes after age 12. Their frequency in healthy people is similar to that of ICA, 3. Antibodies tothe 65-KD isoform of glutamic aad decarboxylase (GADss) have been found up to 10 years before the clinical onset of type I diabetes and are present in ~60% of patients with newly diagnosed diabetes. GADas antibodies have been used to identify patients with apparent type 2 diabetes who will subsequently progress to type I diabetes 4, Two insulinoma associated antigens, LA-2A and 1A-28 (also called 1A-2BA), are directed against two tyrosine phosphatases. They have been detected in more than. 50% of newly diagnosed type I diabetes patients. Genetics, Susceptibility to type 1 diabetes is inherited, but the mode of inheritance is complex and has not been completely defined Ieis a multigenie wait, and the major locus is the major histo- ‘compatibility complex on chromosome 6, At least LI other loci on 9 chromasomes also contribute, with the regulatory region of the insulin gene on chromosome [ip15 being an important locus. The concordance rate between identical twins is approximately 30%, and approximately 95% of whites with type | diabetes express either human leukocyte antigen (HLA)-DR3 or HLA-DR‘ histocompotibilty antigens, How- ever, up to 40% of the nondiabetic population also express these alleles. In contrast, the HLA-DQB1*060? allele signifi cantly decreases the risk of type I diabetes. FLA typing can indicate absolute risk of diabetes. The risk ofa sibling develop- ing diabetes is 1%, 5%, and 10% to 20% if the number of haplotypes shared is none, one, and two, respectively. How- ever, only 10% of patients with type 1 diabetes have an affected first-degree relative. The multiplicity of independent chromosomal regions associated with a predisposition to type 1 diabetes suygests that other suscepribility genes will be identified. Routine measuremenc of genetic markers is not of value at this time for the diagnosis or management of patients with type 1 diabetes.” Environment Environmental factors are thought to be involved in initiating diabetes. For example, viruses, such as rubella, mumps, and coxsackievirus B, have been implicated. Other environmental factors that have been suggested include chemicals and cow's milk, Pathogenesis of Type 2 Diabetes Mellitus Insulin resistance and f-cell dysfunction ate pathological defects in patients with type 2 diabetes, Insulin resistance isa decreased382 PART IV Analytes ability of insulin to act on the peripheral tissue and is thought to be the primary underlying pathological process. cell dys function is an inability of the pancreas to produce sufficient insulin «© compensate for the insulin resistance. Clinically, there is a relative deficiency of insulin early in the disease and absolute insulin deficiency late in the disease, It is uncertain whether type 2 diabetes is primnrily due to a defect in B-cell secretion, peripheral resistance <0 insulin, or both. However, there are data to support the concept chat insulin resistance is the primary defect, preceding the derangement in insulin secretion and clinical diabetes by as much as 20 years. Despite the lack of consensus, it is clear that type 2 diabetes mellitus isan extremely heterogeneous disease and that no single cause is adequate to explain the progression from normal glucose tolerance to diabetes. The fundamental molecular defeets in insulin resistance and insulin secretion result feom a combina- tion of genctics and environmental genetic factor. Insulin Resistance Insulin resistance is defined asa decreased biological response to normal concentrations of cncwlatng insulin, It is found in both obese, nondiabetic. individuals and in patients with type 2 diabetes. The underlying pathophysiology has not been icenti- fied, but insulin resistance is usually areributed co a defect in insulin action, Measurement of insulin resistance in a routine clinical setting is dificule, and surrogate measures, such as fasting insulin concentration, are used to provide an indisect assessment of insulin function. There is a broad clinical spec- trum of ingulin resistance, varying from euglycemia (with a romble increase in endogenots insulin) to hyperglyeemia despite lange doses of exogenous insulin. ‘The insulin resistance syndreme (also known as syndrome X or the metabolic syndrome, http/fwmw.americanheart.org) isa constellation of associated clinical and laboratory findings, consisting of (I) insulin resistance, (2) hyperinsulinemia, (3) obesity, (4) dyslipidemia (high tiglyceride and low HDL chor lesterol), and (5) hypertension, The metabolic syndrome is diagnosed ifan indivichal meersthree or more ofthe following criteria * Abslominal obesity: waist citcumference greater than 35 ches (women) oF 40 inches (men) riglycerides greater than 150 mgd. * HDL cholesterol less than 50 mu/aL. (women) or less chan 40 mgldL (men) * Blood pressure greater than o: equal to 130/85 mm Hg ** Fasting plasma glucose (FPG) greater than or equal to 110 mgd Individuals with this syndrome are at increased risk for cardiovascular disease. Several rare clinical sndeomes are also associated with insulin resistance, The prototype is the type A insulin resistance syndrome, which is characterized by (I) hyperinsulinemia, (2) acanthoss nigricans, and (3) ovarian bhyperandogenism. Loss of B-Cell Function The increased cell demand induced by insulin resistance is associated with a. progressive loss of fi-cell function thac is necessary for the development of fasting hyperglycemia ‘The major defect is a loss of ghucose-induced insulin release that is termed selective glucose urvesponsiveness. Hlyperglyeeria appears to render the B-cells increasingly unresponsive to slucose (a condition called glucotoniity), and the extent of dysfunction correlates with both the glucose concentration and the dlaration of hyperglycemia. Restoration of euglycernia rapidly resolves the defect. Oxher insulin secretory abnormali- ties in individuals with type 2 diabetes include disruption of the normal pulsatile release of insulin and an increased. ratio of plasma proinsulin co insulin, Evidence from knockout ‘mice indicates that insulin resistance in the Brcells may con- tribute to alterations in insulin secretion as occurs in type 2 diabetes. Diabetogenes Genetic factors contribute to the development of type 2 diae betes. For example, the concordance rate for type 2 diabetes in identical twins approaches 100%. In addition, ype 2 diabe- tes is 10 times more likely to occur in an obese incividual with ‘a parent who has diabetes than in an equally obese individual srithout a diabetic family history. The mode of inheritance, hhowever, is unknown, and type 2 diabetes has been described as a “geneticis’s nightmare.” For example, it is genetically more complex than mendelian disorders and is not inherited according to mendelian rules. Multiple genetic factors interact with exogenous influences (such as environmental factors) to produce the phenotype. “Multiple factors complicate the search for diahetogenes in type 2 diabetes. A variety of approaches have identified several senes that are associated with type 2 diabetes. However, despite considerable investigative efforts to identify the genetic basis of type 2 diabetes mellitus, genetic defects identified to date account for fewer than 5% of individuals with type 2 diabetes. ‘Therefore the gene or genes eausing the eammon forms of type 2 diabetes remain unknown. The known genes afect insulin secretion, participate in insulin action, or regulate bedy weight. Environment Environmental factor, such as diet and exerese, are important determinants in the pathogenesis of type 2 diabetes. Convine- ing evidence links obesity to the development of type 2 diabe- tes, but the association is complex, Although. 60% to 80% of those with type 2 diabetes are obese, diabetes develops in fewer than 15% of obese individuals. In contrast, vireuely all cbese people, even those with normal carbohydrate tolerance, have hyperinsulinemia and are insulin resistance. Oxher factors, such as (1) family history of type 2 diabetes (genetic predisposition), (2) the duration of obesity, and (3) the distribution of ft, also are important, ‘An inverse relationship exists between the level of physical activity and the prevalence of type 2 diabetes. The risk of type 2 diabetes decreases by 6% for every 500-keal increase in daily energy expenditure. The mechanism of the proteccive effect of ‘exercise is thought to be an increased sensitivity -0 insulin in skeletal muscle and adipose tissue. Diagnosis The diagnosis of diabetes mellitus depends solely on the dem- nstraton of hyperglycemia (Box 22-2). For type I diabezes, the diagnosis is usually easy because hyperglycemia (1) appears abruptly, (2) is severe, and (3) is accompanied by serious met- bolic derangements, Digguosis of ype 2 diaberes may be die ficult because the metabolic changes are often not severe enough for the patient to notice symptoms of them.