Chapter 2 intestinal absorption and bioavailability of vitamins

Intestinal Absorption and Bioavailability of Vitamins: Introduction 2.1 General Principles of Solute Translocation Mammalian epithelia are enveloped by a plasma membrane composed of a ph

Intestinal Absorption and Bioavailability

of Vitamins: Introduction

2.1 General Principles of Solute Translocation

Mammalian epithelia are enveloped by a plasma membrane composed of

a phospholipid bilayer interspersed frequently with cholesterol mol-ecules Integral transmembrane proteins span the lipid bilayer in a weaving fashion and account for most membrane-associated receptors and transporters and certain enzymes Tight junctions prevent the passage of water and molecular solutes between adjacent epithelial cells The plasma membrane constitutes a selective barrier to the transcellular movement of molecules and ions between the extracellular and intra-cellular fluid compartments Fat-soluble substances, water, and small uncharged polar solutes can simply diffuse through the membrane, but ions and water-soluble molecules having five or more carbon atoms cannot do so Most biologically important water-soluble substances (e.g., glucose, amino acids, water-soluble vitamins, and certain inorganic ions) are translocated across the plasma membrane by means of protein transporters, which exert their effect through a change in their three-dimensional shape Specific transporters are responsible for the translocation of a specific molecule or a group of closely related mol-ecules Specificity is imparted by the tertiary and quaternary structures

of the transporter molecule — only if a solute’s spatial configuration fits into the protein, will the solute be transferred across the membrane Transporters fall into two main classes: carriers and ion channels Ion pumps are a type of carrier protein, which is also an enzyme

At physiological concentrations, the translocation of several water-soluble vitamins (thiamin, riboflavin, pantothenic acid, biotin, and vitamin C) across cell membranes is mediated by carrier proteins The term “transport” implies a carrier-mediated translocation The interaction

of a transportable substrate with its carrier is characterized by saturation at high substrate concentration, stereospecificity, and competition with structural analogs These properties are shared by the interaction of a substrate and an enzyme, and therefore, the terms Vmax and Kmcan be

used to describe the kinetics of transport The maximum rate of transport (Vmax) is the point at which all of the available binding sites on the carrier are occupied by substrate — a further increase in the substrate concen-tration has no effect on the transport rate Vmax values are expressed in picomoles of substrate per milligram protein during a specified period

of minutes Each carrier protein has a characteristic binding constant (Km) for its substrate Km is defined as the concentration of substrate (expressed in units of molarity, mM) at which half of the available carrier sites are occupied and is determined experimentally as Vmax/2

Km describes the affinity of the carrier for its substrate in a reciprocal manner and is independent of the amount of carrier The lower the value of Km, the greater the affinity of the carrier for its substrate and the greater the transport rate

The downhill movement of a substance from a region of higher concen-tration to one of lower concenconcen-tration is a passive process driven by the con-centration gradient There are two types of passive movement: facilitated diffusion, which is carrier mediated, and simple diffusion, which is not The uphill movement of a substance is referred to as active transport, either primary or secondary, and requires the expenditure of metabolic energy Primary active transport is driven directly by metabolic energy and

is carried out exclusively by ion pumps, such as the calcium pumps, the sodium pump, and the proton pumps Ion pumps are ATPases, which utilize the energy released by the hydrolysis of ATP Secondary active transport is indirectly linked to metabolic energy through a coupling of the solute to the pump-driven movement of an inorganic ion (usually Naþ)

At many places in the body, substances must be translocated all the way through an epithelium, instead of simply through the plasma membrane Movement of this type occurs, for example, through the epithelia of the intestine and renal tubules The vectorial nature of such movement is made possible by the polarity of the cell surface, whereby distinct sets

of surface components (carriers, ion channels, and ion pumps) are loca-lized to separate plasma membrane domains Transepithelial movement may involve concentrative active transport through the apical membrane domain, and facilitated diffusion for the downhill exit through the baso-lateral membrane domain

2.2 Intestinal Absorption

2.2.1 The Villus

The functional absorptive unit of the small intestine is the villus, a finger-like projection of the mucosa Contained within the lamina propria core of each

villus is a capillary network with a supplying arteriole and draining venule.

A blind-ending lymphatic vessel (lacteal) in the center of each villus drains into a plexus of collecting vesicles in the submucosa Each villus is covered

by an epithelium composed of a single layer of columnar absorptive cells (enterocytes) interspersed occasionally with mucus-secreting goblet cells The enterocyte constitutes the only anatomical barrier of physiological sig-nificance controlling the absorption of nutrients The apical membrane of the enterocyte (i.e., the membrane facing the intestinal lumen) is covered with microvilli, which are minute projections of the plasma membrane Because of its brush-like appearance under the microcope, the apical mem-brane is also known as the brush-border memmem-brane

2.2.2 The Luminal Environment

Bulk contents of the intestinal lumen are mixed by segmentation and peri-stalsis, and water and solutes are brought to the surface of the mucosa by convection However, the luminal environment immediately adjacent to the brush-border membrane is stationary and unaffected by gut motility The lack of convective mixing in this region creates a series of thin layers, each progressively more stirred, extending from the surface of the enterocyte to the bulk phase of the lumen These thin layers constitute the so-called “unstirred layer,” whose effective thickness has been calculated to be 35 mm [1]

Solute movement within an unstirred layer takes place by diffusion, which is slow compared with the convective movement in the bulk luminal phase The pH at the luminal surface is approximately two units lower than that of the bulk phase and varies less than+0.5 units, despite large pH variations in the intestinal chyme It has been suggested that the formation of the low-pH microclimate is due to the presence

of mucin, which covers the entire surface of the epithelium [2,3] Mucopolysaccharides possess a wide range of ionizable groups and hence mucin is an ampholyte If the luminal chyme is of low pH, the ampholyte

is positively charged, and so it repels additional hydrogen ions entering the microclimate If, on the other hand, the chyme is alkaline, the ampholyte becomes negatively charged, and retains hydrogen ions within the micro-climate In this manner, the mucin layer functions as a restrictive barrier for hydrogen ions diffusing in and out of the microclimate

2.2.3 Adaptive Regulation of Intestinal Nutrient Transport

2.2.3.1 Nonspecific Anatomical Adaptations to Changing Metabolic

Requirements and Food Deprivation

Increases in metabolic requirements, such as arise during pregnancy, lactation, growth, exercise, and cold stress, are met by an increased Vitamins in Foods: Analysis, Bioavailability, and Stability 25

absorption of all available nutrients, mediated at least in part by an induced increase in food intake The increased absorption is due to an increase in mucosal mass per unit length of intestine and a consequent increase in absorptive surface area Not only is there an increase in the total number of cells, but the villi become taller

The mammalian intestine adapts to prolonged food deprivation by dramatically slowing the rate of epithelial cell production in the crypts in order to conserve proteins and biosynthetic energy This effect on mitosis and enterocyte renewal leads to markedly shortened villi Because cell migration along the crypt-villus unit is also slowed, more cells lining the villi are functionally mature Therefore, food deprivation, by reducing mucosal mass and increasing the ratio of transporting to nontransporting cells, effectively increases solute transport per unit mass of intestine

2.2.3.2 Dietary Regulation of Intestinal Nutrient Carriers

It is well established that certain intestinal nutrient carriers (e.g., those transporting glucose and amino acids) are adaptively regulated by their substrates In response to a signal for regulation of transport, the number of carriers at both the apical and basolateral membranes of enter-ocytes is increased or decreased as appropriate According to Karasov’s adaptive modulation hypothesis [4], a carrier should be repressed when its biosynthetic and maintenance costs exceed the benefits it provides The benefits can be provision of either metabolizable calories or an

“essential” nutrient, that is, a nutrient which cannot be synthesized

by the body and must be obtained from the diet Glucose carriers are up-regulated when the dietary supply of glucose is adequate or high because glucose provides valuable calories The down-regulation of glucose carriers during a deficiency of glucose can be explained

by the biosynthetic and maintenance costs outweighing the benefits of transporting this “nonessential” nutrient

One might expect carriers for water-soluble vitamins to be down-regulated by their substrates and up-down-regulated in deficiency of the vita-mins The rationale in this case is that carriers for these essential nutrients are most needed at low dietary substrate levels; at high levels, the required amount of the vitamin could be extracted from the lumen

by fewer carriers, or even cross the enterocyte by simple diffusion As vitamins do not provide metabolizable energy, there is nothing to gain from the cost of synthesizing and maintaining carriers when the vitamin supply is adequate or in excess

The prediction of suppressed transport of vitamins at high dietary intakes has proved to be true for ascorbic acid, biotin, and thiamin, but not for pantothenic acid, for which carrier activity is independent of dietary levels [5] It appears that intestinal carriers are regulated only if

they make the dominant contribution to uptake, as is the case for the three regulated vitamins It can also be reasoned that carriers for ascorbic acid, biotin, and thiamin would need to be regulated, because nutritional deficiencies of these vitamins can and do occur In contrast, there is no need to regulate pantothenic acid carriers, because this vitamin is found naturally in almost all foods, and cases of deficiency are very rare

2.2.4 Digestion, Absorption, and Transport of Dietary Fat

Absorption of the fat-soluble vitamins takes place mainly in the proximal jejunum and depends on the proper functioning of the digestion and absorption of dietary fat The stomach is the major site for emulsification

of fat The coarse lipid emulsion, on entering the duodenum, is emulsified into smaller globules by the detergent action of bile Pancreatic lipase hydrolyses triglycerides at the 1 and 3 positions, yielding 2-monoglycer-ides and free fatty acids During their detergent action, bile salts exist as individual molecules Above a critical concentration of bile salts, the bile constituents (bile salts, phospholipids, and cholesterol) form aggre-gates called micelles, in which the polar ends of the molecules are orien-tated toward the surface and the nonpolar portion forms the interior The 2-monoglycerides and free fatty acids are sufficiently polar to combine with the micelles to form mixed micelles These are stable water-soluble structures, which can dissolve fat-soluble vitamins and other hydrophobic compounds in their oily interior

Mixed micelles do not cross the brush-border membrane of enterocytes

as intact structures: the products of lipolysis must dissociate from these structures before they can be absorbed Shiau and Levine [6] showed that a low-pH microclimate, representing the unstirred layer lining the luminal surface of the jejunum, facilitates micellar dissociation Presum-ably, the fatty acid components of the mixed micelles become protonated when the mixed micelles enter the unstirred layer This protonation reduces fatty acid solubility in the mixed micelles, allowing release of the fatty acids together with other lipid constituents Individual lipids, including fat-soluble vitamins, can then be passively absorbed across the brush-border membrane The bile salts are left behind to be actively reabsorbed in the distal ileum, whence they return to the liver to be recycled via the gall-bladder

After the lipolytic micellar products enter the enterocytes, a cytosolic fatty acid-binding protein (FABP) facilitates intracellular transport of fatty acids by directing them from the cell membrane to the smooth endo-plasmic reticulum, where triglyceride synthesis takes place The triglycer-ides are packaged into chylomicrons, together with free and esterified cholesterol, phospholipids, apolipoproteins, fat-soluble vitamins, and Vitamins in Foods: Analysis, Bioavailability, and Stability 27

carotenoids After further processing, the chylomicrons are discharged from the enterocyte by exocytosis across the basolateral membrane and enter the central lacteal of the villus From there, they pass into the larger lymphatic channels draining the intestine, into the thoracic duct, and ultimately into the systemic circulation

Medium-chain triglycerides, which contain fatty acids with a chain length of 6– 12 carbon atoms, are not found in appreciable amounts in the normal diet However, they deserve mention because they are included in specialized diets for patients who have fat malabsorption Medium-chain triglycerides are absorbed in a more efficient manner to that described above for the longer-chain triglycerides Being water-soluble, they can be absorbed directly as intact triglycerides Once inside the enterocyte, they are hydrolyzed to medium-chain fatty acids by specific cellular lipases Medium-chain fatty acids do not bind to FABP, are not reesterified to triglycerides, and are not packaged in chylomicrons After leaving the enterocyte, medium-chain fatty acids enter the portal vein where they are bound to albumin and transported to the liver [7]

The chylomicrons are carried by the blood to all the tissues Associ-ated with the endothelium of blood capillaries in most tissues is the enzyme lipoprotein lipase, which attacks circulating chylomicrons and converts them into much smaller triglyceride-depleted particles known as chylomicron remnants These particles contain apolipo-protein E (apoE) acquired from other circulating lipoapolipo-proteins The released free fatty acids and diglycerides can then be absorbed by the tissue cells

The liver has the capacity to rapidly remove chylomicron remnants from the circulation, the apoE on the remnants serving as the ligand for receptors present on the surface of hepatocytes The fates of individual fat-soluble vitamins after liver uptake of chylomicron remnants are

dis-2.2.5 Transport of Glucose and Fructose: A Model for the Absorption

of Some Water-Soluble Vitamins

Glucose and fructose transport have been well studied [8], and the experimental techniques and postulated mechanisms help toward under-standing the absorption of water-soluble vitamins

are absorbed by the small intestine Luminal glucose crosses the epithelial brush border and accumulates in the enterocyte by means of secondary active transport Transport is mediated by a sodium– glucose cotransport-ing carrier (SGLT1), which binds the substrates at a stoichiometric ratio of cussed in their respective chapters (3– 6)

Figure 2.1shows how physiological amounts of glucose and fructose

two sodium ions to one glucose molecule The immediate driving force for the sodium-coupled entry of glucose is the electrochemical gradient for sodium This has two components: an electrical potential difference

of about 40 mV across the brush-border membrane (cell interior negative) and a sodium concentration gradient Both the electrical and chemical

Na+

Na +

Na +

Na +

LUMEN

brush-border

membrane

tight junction

basolateral membrane

SEROSA

intercellular space

GLUT2

GLUT2

GLUT5

glucose

glucose

SGLT1

K+

K+

FIGURE 2.1

The carrier-mediated transport of D -glucose and D -fructose across the apical membrane and basolateral membrane of an enterocyte Naþextruded into the intercellular space by the basolateral Naþ–Kþ-ATPase (sodium pump) is able to equilibrate with Naþ on the luminal side of the enterocyte by permeation through the tight junction ATP, adenosine triphosphate; ADP, adenosine diphosphate; Pi, inorganic phosphate (From Ball, G.F.M., Vitamins Their Role in the Human Body, Blackwell Publishing Limited, Oxford, 2004, p 12 With permission.)

Vitamins in Foods: Analysis, Bioavailability, and Stability 29

components are established by the constant extrusion of sodium out of the enterocyte by the action of the basolateral sodium pump Fructose crosses the brush border by facilitated diffusion mediated by the glucose trans-porter GLUT5 Exit of both glucose and fructose from the enterocyte to the serosa takes place by facilitated diffusion at the basolateral membrane and is mediated by GLUT2

2.2.6 Effects of Dietary Fiber on Absorption of Nutrients

Dietary fiber consists of plant material that cannot be digested by the endogenous secretions of the human digestive tract From an analytical standpoint, dietary fiber can be divided into insoluble fibers and soluble fibers The insoluble fibers include cellulose, lignin, and many hemicelluloses; the soluble fibers include pectins, some hemicelluloses, gums, and mucilages The various gums and mucilages are widely used in the food and pharmaceutical industries as emulsifiers, thickeners, and stabilizers The nature and physical properties of the main fiber Vahouny and Cassidy [10] discussed potential mechanisms by which dietary fiber can modify nutrient absorption Intestinal absorption of nutrients can be influenced by modifying the rates at which food enters

or leaves the stomach Bulky, high fiber foods may require longer periods for ingestion, and therefore modify rates of gastric filling Viscous fiber components slow stomach emptying The delayed release

of gastric emptying and modified intestinal pH might alter the regulation

of pancreatic and biliary secretions Insoluble fibers accelerate small intes-tinal transit, allowing less time for nutrient absorption; in contrast, viscous fibers slow transit Many fiber components can alter the activities

of pancreatic enzymes by affecting viscosity and pH, and by adsorption Dietary fiber impairs lipid absorption by interfering with micelle for-mation Evidence is the in vitro binding of bile salts and other micellar components by lignin and guar gum, and the increase in fecal bile salts

in response to ingestion of dietary fiber Viscous fibers can influence nutri-ent absorption by interfering with bulk phase diffusion of nutrinutri-ents in the intestinal lumen The mucin layer covering the mucosal surface has been suggested to be an important diffusion barrier to absorption Reported changes in mucin content or turnover in response to various fiber types

is a possible mechanism by which dietary fiber alters the transport charac-teristics of nutrients at the mucosal surface Prolonged feeding of diets supplemented with cellulose or pectin significantly increased villus height and thickness, thereby increasing the absorptive surface area The dietary supplements also improved nutrient uptake by the small intestine in vitro

components are summarized inTable 2.1[9]

TABLE 2.1

Dietary Fiber Components

Cellulose Linear polymer of glucose with beta

1– 4 linkages

component of plant cell wall

Binding of water

Lignin Highly complex nonpolysaccharide

polymer derived from phenolics

of woody plants

Binding of bile salts and other organic material Hemicelluloses Heterogeneous group of

polysaccharides which contain a variety of different sugars in the polymeric backbone and side chains

Many insoluble, some soluble Matrix of plant cell wall Binding of water and

cations

Pectins Polymer composed primarily of

galacturonic acid and rhamnose with a variable degree of methyl esterification

Soluble, capable of forming gels with sugar and acid

Matrix of plant cell wall, ripe fruits

Formation of gels, binding of bile salts and other organic material Gums Complex group of highly branched

polysaccharides (e.g., gum acacia)

Soluble to give very viscous colloidal solutions

Extruded at site of injury to plants

Similar to pectins Mucilages Polysaccharides resembling

hemicelluloses (e.g., guar gum)

Soluble to give slimy, colloidal solutions

Mixed with starch in endosperm

Binding of water, formation of gels, binding of bile salts and other organic material Source: From Anderson, J.W and Chen, W.-J.L., Am J Clin Nutr., 32, 346, 1979 With permission.

© 2006 by Taylor & Francis Group, LLC

2.3 Bioavailability

2.3.1 General Concepts

The term “bioavailability,” as applied to food-borne vitamins in human nutrition, refers to the proportion of the quantity of vitamin ingested that undergoes intestinal absorption and utilization by the body Utilization encompasses transport of the absorbed vitamin to the tissues, cellular uptake, and ultimate fate of the vitamin The latter can

be conversion to a form that can fulfill some biochemical or physiological function, conversion to a nonfunctional form for subsequent excretion, and storage The major component of bioavailability and the rate-limiting factor is absorption Many ways of determining vitamin bioavailability have been reported, most of which give an estimate of relative rather than absolute bioavailability Relative bioavailability is commonly expressed as a percentage of the response obtained with a reference material of high bioavailability Bioavailability is an operational term defined by the method used to determine it Different values will be obtained within a given study if different endpoints are used

Intestinal absorption, and therefore bioavailability, of a vitamin depends on the chemical form and physical state in which the vitamin exists within the food matrix These properties may be influenced by the effects of food processing and cooking, particularly in the case of pro-vitamin A carotenoids, niacin, pro-vitamin B6, and folate The food matrix enhances vitamin absorption by stimulating the secretion of digestive enzymes and bile salts Bile salts inhibit gastric emptying and proximal intestinal transit, resulting in an increased residence time at the absorp-tion sites Thus, absorpabsorp-tion of a riboflavin supplement taken with a meal was about 60%, as compared to 15% on an empty stomach [11]

In foods derived from animal and plant tissues, the B-group vitamins occur as their coenzyme derivatives, usually associated with their protein apoenzyme In addition, niacin in cereals and vitamin B6 in certain fruits and vegetables occur largely as bound storage forms In milk and eggs, which are derived from animal secretions, the B-group vitamins occur, at least to some extent, in the underivatized form, a pro-portion of which may be associated with specific binding proteins Vita-mins that exist as chemically bound complexes with some other material in the food matrix exhibit lower efficiencies of digestion and absorption compared with the free (unbound) vitamin ingested, for example, in tablet form

Certain dietary components can retard or enhance a vitamin’s absorp-tion, therefore the composition of the diet is an important factor in bioavailability For example, the presence of adequate amounts of