Digestion and Absorption of Minerals I

(Unifying principles that apply to all minerals)

Digestion

• Preparing for absorption

• Liberating minerals from a bound state to an aqueous phase

• Digestive enzymes

• Bile acids and salts that work with digestive enzymes (e.g., lipases)

Purpose of digestion to mineral nutrition

Minerals in a food source are locked within a matrix composed primarily of proteins, complex carbohydrates and fats

The purpose of digestion is to render large composite molecules into smaller manageable units…minerals are liberated during this process

Digestive processes consists mainly of hydrolytic enzymes that break chemical bonds between modular units without total destruction (metabolism) of the liberated components

Products of the digestate aid in the solubilization and absorption of minerals

Digestive Enzymes (hydrolases)

Enzyme Location Target Action

Pepsin gastric juice proteins breaks peptide bonds

Trypsin and chymotrypsin

duodenum proteins breaks peptide bonds

Amylases saliva and duodenum

starch and glycogen

breaks glycosidic bonds

Lipases duodenum complex lipids

breaks ester bonds

Glycosidases microvilli di- and tri- saccharides

breaks glycosidic bonds

Peptidases microvilli small peptides

breaks peptide bonds

I

II

Phase I is primarily salivary and pancreatic secretions

Phase II involves enzymes on the surface of absorbing cells

Critical factors in Mineral Absorption

• Absorption tends to be selective for the mineral• (makes finding a unified mechanism more difficult) • A deficiency increases the fraction of that mineral absorbed• (absorption is tuned to internal bodily needs)• Certain food chemicals (e.g., phytate, oxalate) lower

absorption by tying up the mineral • There is competition for absorption machinery• Metal ions antagonism (Cu-Zn; Zn-Fe; etc.) occurs at ion

channels during the transmural passage phase of absorption

• Vitamin dependency is seen with Vitamin D and C that regulate body load of Ca+2 and Fe2+respectively

• Absorptive cells excrete factors that aid in the solubility of metal ions

• Some transport proteins are in vesicles that fuse with the membrane and move vectorially within the cell

Steps in mineral absorption

1. Transport through the luminal (apical) cell membrane, i.e., start of transcellular

2. Handling within the enterocyte, i.e., mediate transcellular

3. Transport through the antiluminal basolateral membrane into the circulation, i.e., end of transcellular.

Only metals in an aqueous phase can be transported into the enterocyte

4. Transport between the cells, i.e., paracellular

Two categories of ingested metal Ions

1. Solubility not dependent on pH

2. Solubility pH dependent

Examples: Na+, K+, Mg2+, Ca2+

Examples: Cu2+, Fe2+, Mn2+, Zn2+

Category 2 metal ions are soluble in acid, but form insoluble hydroxy-polymers at neutral or alkaline pH.

Category 1 metal ions are soluble throughout the gastrointestinal pH range (1-8)

Solubility and Metal Ion Absorption

Mucosal Side

Serosal Side

BasolateralSurface

(antiluminal surface)

Apical surface

Microvilli

Ca Ca Ca

Enterocyte

To access the serosal side, the mineral must

pass either through the enterocyte (transcellular

99%) or the junction between enterocytes (paracellular <1%))

Fe Fe

Fe A large fraction of the iron can be trapped (sequestered) within

the cytosol of the enterocye)

Role of Vesicles in the Regulation of Mineral Absorption

Vesicles are internal membrane compartments that move between the cytosol and membranes. This movement is regulated by external factors

Vesicles contain the transport proteins that absorb the mineral into the lumen of the vesicle and bring it into the cell

Vesicles that have fused with the membrane are positioned to absorb minerals. Absorption thus depends on the number of vesicles that fused with the membrane.

Resting Cell Absorbing Cell

MACROMINERALSMonovalent cations, Na+, K+

Monovalent anions, Cl-

Divalent cations, Ca2+, Mg2+

Complexes, HPO4=, HCO3

-

Rule 1: Macrominerals in general enter intestinal cells through transport portals designated for the mineral (major) or between cells (minor).

Rule 2: The energy for entry is provided by a concentration gradient across the membrane or by hydrolysis of ATP (active transport)

Rule 3: Electroneutrality is sought in the operation of membrane co-transporters

Rules that apply to the absorption of Macrominerals

Macrominerals

Na+, K+, Cl-, HPO4-, Mg2+, Ca2+

The macrominerals for the most part rely on diffusion controlled mechanisms combined with specific channel proteins to pass into the system.

Gradients across the membrane can be driven by unidirectional and bidirectional ATPase enzymes

Example

Na+/K+ ATPase

Ca2+/H+ ATPase

Properties of Macrominerals Relative to Absorption

2. Monovalent ions are unable to form stable complexes

1. Monovalent ions exist mostly as free ions

3. Divalent ions exist partially as free ions

4. Divalent ions are more apt to form complexes with proteins and organics

5. Complexes exist mainly as free ions

Absorption of Sodium and Chloride

Na+

Na+

Glucose

Amino acids

H+

Cl-

HCO3-

Apical (lumen) side

Glucose cotransporter

Amino acid transporter

Na+/H+ antitporter

CO2 CO2

H2CO3

H+

H+ + HCO3-

H2O

2K+

3Na+

Anion antiporter

Blood

Intestinal Enterocyte

Carbonic anhydrase

ATPase

Na+/K+ ATPase

Calcium and Magnesium

Mucosal surface [import] (channel proteins, ATPase enzymes, reductases)

Cytosol [storage] (transport and storage proteins, vesicles)

Serosal surface [export]

Microvilli

Three stages in intestinal absorption at the cellular level

Calcium absorption is the sum of saturable and unsaturable processes

1. Solubility depends on dietary source

2. CaHPO4 is 18 time more soluble than CaCO3

3. Solubility also depends on pH

4. Transcellular and paracellular transport processes

5. Transcellular proximal intestine saturable, regulated

6. Paracellular throughout intestine unsaturable, unregulated

7. Vitamin D is the major regulator of transcellular calcium entry

8. Calcium channels in brush border and apical membranes appear to have a vitamin D-sensitive element

Inverted sac

Ca

CaCa

Ca Ca

Everted Sac and Intestinal Loop Technique to measure Ca2+ Absorption

Transcellular movement of Ca2+ into the sac is a metabolically active process requiring oxygen and occurs against a concentration gradient.

In situ Intestinal Loop

45Ca45Ca

45Ca

Absorption is the sum of two processes: saturable and non-saturable

Ca

Ca

CaCa

Absorption is the amount of Ca2+ effusing with time as measured at

different concentrations of Ca2+

IntestineCat1

Calbindin

Liver

Kidney

Ca2+

Bone

Parathyroid

PTH1,25-OH D3

(Calcitriol)

25-OH D3

Ca3(PO4)2

PTH

Serum

Ca2+

Cholecalciferol

Resorption

Decrease Excretion

Activate transcription of Cat1 and calbindin

Activate hydroxylase

Activate osteoclasts

Ca2+

Calcitriol

Saturable

Total

Unsaturable

In situ intestinal loop experiment showing Ca2+ absorption cannot be due to simple diffusion, but is the sum of two

processes, saturated and unsaturated

Time

%Abs

1 mM

25 mM

100

200 mM

100 mM

50

10 mM

% absorbed = % of total sac 45Ca that effused out

Total = sum of saturated and unsaturated at each time point

50

100

00 100 200

Dietary Calcium

CalciumAbsorbed

50

100

00 100 200

Dietary Calcium

-Vit D - Vit D + 1,25-(OH)2-D3

Saturable

Non-Saturable

Duodenum Vitamin D deficient rats

00 100 200

50

100

00 100 200

Saturable

00 100 200

Saturable

Non-saturable

Duodenum Jejunum Ileum

Calcium Instilled, mMUptake in ileum is by diffusion only; it is, therefore, not regulated by vitamin D. Thus, most of the Ca2+ is absorbed in the duodenum.

Ficks Law of diffusion: The rate of diffusion of an ion at steady-state transmembrane flux varies inversely with path length and directly with area and concentration gradient

F = ADca

L([Ca]1 – [Ca]2)

A = 80 m2

L = 10 m

Dca = 3 x 10-3 cm2/min

Based on Fick’s law, the expected diffusion rate of Ca across the intestinal cell is 96 x 10-18 mol/min/cell.

Adolph Fick

Rate observed in the laboratory is 70 times greater at Vmax, which means duodenal cells have factors that enhance self diffusion of Ca

Possible factor is Calbindin, a small (9 kD) Ca-binding protein

after Bronner

Search for the Vitamin D sensitive Factor

1. Calbindin (9 kd cytosolic Ca-binding protein)2. CaT1 (a calcium channel protein in brush border of intestinal cells)

1,25 dihydroxy vitamin D3 given at time 0 increases the expression of CaT1

Changes in CaT1 mRNA levels with different amounts of D3

CaT1, a Ca channel protein in the brush border of human enterocyte, is regulated by 1,25-dihydroxyvitamin D. The vitamin appears to mediate changes in CaT1-mRNA levels. CaT1, therefore, could be the primary gatekeeper regulating homeostatic modulation of intestinal calcium absorption efficiency.

Take Home

Our best understanding is that calcium enters the duodenal cell through calcium channels which may contain a vitamin D responsive Ca-binding component. Entry is down an electrochemical gradient.

Bonner, 1999

Ca2+Ca2+

Calbindin

ATPase

ATPase

Ca2+

CAT1

Mg2+(Na+)

Ca2+

Calcium ATPaseEnterocyte

ParacellularCa2+

Ca bound to fiber, phytate, oxalate, fatty acids

Calcium ATPaseantiporter

Lumen Blood

Ca2+

Calcium Absorption

Albumin

Vitamin D responsive

CAT1 is a Ca2+ channel protein located in the brush border of mucosal cells

Calbindin is a small (9 kD) protein in the cytosol of mucosal cells

Unanswered Questions

1. Where exactly is CaT1 located and does raising CaT1 protein require it relocation to the absorbing membrane?

2. Is there any evidence for CaT1 location in mobile vesicles?

3. Does 1,25-dihydroxy vitamin D3 affect efflux of Ca2+ at the basolateral surface?

4. Does CaT1 also recognize Mg2+?

Phosphorus (phosphate)

Phosphorous

Phosphorous absorption utilizes a Na/phosphate cotransporter (Npt2a)

1. Expressed in the brush border membrane

PO4=

Duodenum, Jejunum

Na+

Npt2a

(Ca2+, Mg2+)

PO4=

Enterocyte

Saturable, carrier-mediated

PO4=

Complexed with other minerals or as organic

phosphateVitamin D stimulated

3. non-regulated diffusion may be the major absorption pathway with higher intake

2. Saturable, carrier mediated and responsive to Vit D.

Magnesium1. Absorption depends on concentration

2. Absorption is saturable and non-saturable (7-10%)

3. Fully saturable in ileum but not jejunum (contrast with calcium)

5. Vitamin D has no influence on magnesium absorption

4. Absorption in the colon significant

Human Study

Fed Fractional Absorption

7 mg 65-75%

36 mg 11-14%

Magnesium

Enterocyte

Mg2+

TRPM6

Distal jejunum and ileum

Cation channel protein (transient receptor protein TRP)

Mg2+

Since TRPM6 operates by diffusion without co-transporters, Mg2+

absorption efficiency depends on the amount of Mg2+ in the diet and within

the cell

ATPase

ATP

ADP

Mg2+

Mg2+ -bound to phytate, fiber, fatty acids

Microminerals3d metals: Fe, Zn, Cu

Microminerals

Because of their very low cellular concentrations, the micronutrients rely on specific high affinity transporters and binding proteins for movement. Some collect in vesicles and use the vesicle as the transport factor.

Fe2+, Cu2+, Mn2+, Zn2+

Redox-sensitive metals (Fe2+/Fe3+, Cu+/Cu2+) rely on valence state changes to be sequestered or transported from the cell.

Metals such as Fe3+ and Zn2+ are more soluble in acid solutions due to a shift in the equilibrium towards the free ion

Fe(OH)3(s) Fe3+(aq) + 3OH-(aq)

H+Zn(OH)2(s) Zn2+(aq) + 2OH-(aq)

Pulls equilibria

pH

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Solubility Fe(OH)3 solubility

Zn(OH)2 solubility

Elements of Micromineral Absorption

• Insolubility or iron and zinc is partially overcome by mucins secreted from the cells

• Only Fe3+ and Cu+ can engage their respective transporters

• Cytosolic sequestering and regulatory factors have the potential to lock the mineral within the cell and block its release

• Internal movement of Zn2+, Cu+ and Fe3+ is primarily via vesicles

• Basolateral surface release is redox sensitive for Fe and Cu

• See Powell et al. The regulation of mineral absorption in the gastrointestinal tract. Proc. Nutr. Soc. 58(1), 147-153 (1999)

Mucins are complex polysaccharides secreted into the lumenthat assist in stabilizing the solubility of metal ions

Mucins prevent alkaline-induced polymerization of category 2 metal ions and make the metal ion available to transporters on the

enterocyte surface

Mucins

Correlation of spectra of Fe with iron absorption

Importance of mucins in making “insoluble”

iron available to membrane transporters

Rudzki et al, 1973Conrad et al, 1991 as cited in Powell et al, 1999

Laminated mucous layer

Stomach (pyloric mucosa) Intestine (colon)

Mucous layer

Pyloric mucosal

cells

Mucosal goblet cells

Aluminum localization with the mucous layer at rat villi surfaces

Events in the Cellular Absorption of Iron

Heme Iron

Non-heme iron

Ferric (Fe3+) Iron Pathway

Ferrous (Fe2+) Iron Pathway

Three Pathways in Iron Absorption

Fe3+ PathwayMobilferrin-integrin

Fe2+ PathwayDivalent cation transporter (DCT-1, DCM-1,Nramp2)

Heme Pathway

DCT1Fe3+

Mobilferrin

Ferroportin 1

Mobilferrin-Fe3+

Hephaestin

Fe2+Dctyb reductase

CuCuCu

Heme carrier protein

integrin

Fe2+

Porphyrin ring

gastroferrin

Duodenal Lumen Duodenal Mucosa Plasma

Heme-Protein

Heme+

Polypeptides

Mucin(gastroferrin)

Fe3+

Fe3+ Fe3+

B2-microglobulin

HFE Biliverdin Bilirubin Bilirubin

CO COHeme

Oxygenase

Heme

FeFe2+2+

FeFe33

++

DCT-1

B3 integrin

paraferrin

Mobilferrin (vesicles)

FeFe2+2+ FeFe2+2+

FeFe33

++Transferrin

Iron Absorption (heme and non-heme)

Ferroportin

Ferritin

FeFe3+3+

FR FeFe3+3+Hephaestin

Dcytb reductase

Nramp2 (Natural resistance associated macrophage protein)

Nramp1 Nramp2

Nramp2

(no iron transport)

(DMT1/DCT1)

Transport Mn2+,Fe2+, Ni2+

DMT1 isoform 1 DMT1 isoform 2

Secretions into the lumin (soluble mucins) retard hydrolysis of Cu, Fe and Zn permitting binding to transporters and more efficient uptake.

Soluble mucins (gastroferrin)

Efficiency of transport is related to valance state with M+ > M2+ > M3+

Redox-active factors reduce Fe3+ to Fe2+

Fe3+ reductase

Divalent cation transporter (DCT1) transports M2+ metals (Fe2+, Ca2+,Cu2+, Zn2+), keeping out toxic metals such as Al3+. A former name of DCT1 is Nramp2.

DCT1FeR

Mobilferrin

Mobileferrin on the inner side of the apical membrane receives metal from DCT1 and transfers it to cytosol.

Integrin anchor

Mobilferrin

Ferritin (or paraferritin) or Fe

2-microglobulin (for Zn)

HFE (human leukocyte antigen H)

HFE may be involved in stabilizing the above complexes to mobiltransferrin