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
I
II
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 tribreaks glycosidic bonds
saccharides
Peptidases
microvilli
small
peptides
breaks peptide bonds
Phase I is primarily salivary and pancreatic secretions
Phase II involves enzymes on the surface of absorbing cells
Critical factors in Mineral Absorption
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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.
4. Transport between the cells, i.e., paracellular
Only metals in an aqueous phase can be
transported into the enterocyte
Solubility and Metal Ion Absorption
Two categories of ingested metal Ions
1. Solubility not dependent on pH
Examples: Na+, K+, Mg2+, Ca2+
2. Solubility pH dependent
Examples: Cu2+, Fe2+, Mn2+, Zn2+
Category 1 metal ions are soluble throughout the gastrointestinal pH range (1-8)
Category 2 metal ions are soluble in acid, but form insoluble hydroxypolymers at neutral or alkaline pH.
Mucosal Side
Fe
Fe
Ca
Fe
Ca
A large fraction of the
iron can be trapped
(sequestered) within
the cytosol of the
enterocye)
Ca
Microvilli
Apical
surface
Enterocyte
Basolateral
Surface
(antiluminal
surface)
Serosal Side
To access the serosal
side, the mineral must
pass either through the
enterocyte (transcellular
99%) or the junction
between enterocytes
(paracellular <1%))
Role of Vesicles in the Regulation of Mineral Absorption
Resting Cell
Absorbing Cell
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.
MACROMINERALS
Monovalent cations, Na+, K+
Monovalent anions, Cl-
Divalent cations, Ca2+, Mg2+
Complexes, HPO4=, HCO3-
Rules that apply to the absorption of Macrominerals
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
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
1. Monovalent ions exist mostly as free ions
2. Monovalent ions are unable to form stable complexes
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
Blood
Apical (lumen) side
Na+
Glucose
Glucose cotransporter
Amino acids
Amino acid transporter
Na+/K+ ATPase
3Na+
2K+
ATP
ase
Na+
H+
Na+/H+ antitporter
H+
Carbonic anhydrase
H+ + HCO3-
H2CO3
CO2
CO2
H2O
Cl-
Intestinal Enterocyte
HCO3Anion antiporter
Calcium and Magnesium
Three stages in intestinal absorption at the cellular level
Microvilli
Mucosal surface [import] (channel proteins,
ATPase enzymes, reductases)
Cytosol [storage] (transport and storage proteins,
vesicles)
Serosal surface [export]
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
Everted Sac and Intestinal Loop Technique to measure Ca2+ Absorption
In situ Intestinal Loop
Inverted sac
Ca
Ca
Ca
Ca
45Ca
45Ca
45Ca
Ca
Ca
Ca
Ca
Ca
Absorption is the amount of Ca2+
effusing with time as measured at
different concentrations of Ca2+
Transcellular movement of Ca2+ into the sac is a metabolically active process
requiring oxygen and occurs against a concentration gradient.
Absorption is the sum of two processes: saturable and non-saturable
Ca2+
Intestine
Cat1
Calbindin
Activate transcription of
Cat1 and calbindin
Calcitriol
Resorption
Ca3(PO4)2
Bone
Activate
osteoclasts
Serum
Ca2+
PTH
Decrease
Excretion
Activate
hydroxylase 1,25-OH D3
Ca2+
(Calcitriol)
Kidney
25-OH D3
Parathyroid
PTH
Cholecalciferol
Liver
In situ intestinal loop experiment showing Ca2+ absorption
cannot be due to simple diffusion, but is the sum of two
processes, saturated and unsaturated
% absorbed = % of total sac 45Ca that effused out
1 mM
10 mM
100
%
Abs
Total
25 mM
100 mM
Unsaturable
50
200 mM
Time
Saturable
Total = sum of saturated and
unsaturated at each time point
Vitamin D deficient rats
Duodenum
-Vit D
- Vit D + 1,25-(OH)2-D3
100
100
Calcium
Absorbed
Non-Saturable
50
50
Saturable
0
0
0
100
200
Dietary Calcium
0
100
200
Dietary Calcium
Duodenum
Jejunum
Ileum
100
Non-saturable
50
Saturable
0
Saturable
0
0
100
200
0
0
100
200
0
100
Calcium Instilled, mM
Uptake in ileum is by diffusion only; it is, therefore, not regulated by vitamin
D. Thus, most of the Ca2+ is absorbed in the duodenum.
200
Ficks Law of diffusion: The rate of diffusion of an ion at steadystate 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
after Bronner
Adolph Fick
Based on Fick’s law, the expected diffusion rate of Ca
across the intestinal cell is 96 x 10-18 mol/min/cell.
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
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
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
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.
Calcium Absorption
Blood
Lumen
Vitamin D
responsive
Calbindin
CAT1
Ca2+
Calcium ATPase
Enterocyte
ATP
ase
Ca2+
Ca2+
Ca2+
Calcium ATPase
antiporter
ATP
Ca2+
ase
Mg2+
(Na+)
Albumin
Paracellular
Ca2+
Ca bound to
fiber, phytate,
oxalate, fatty
acids
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
2. Saturable, carrier mediated and responsive to Vit D.
3. non-regulated diffusion may be the major absorption
pathway with higher intake
Saturable, carrier-mediated
Duodenum, Jejunum
Npt2a
PO4=
Na+
PO4=
PO4=
(Ca2+, Mg2+)
Vitamin D
stimulated
Enterocyte
Complexed with other
minerals or as organic
phosphate
Magnesium
1. Absorption depends on concentration
Human Study
Fed
7 mg
36 mg
Fractional Absorption
65-75%
11-14%
2. Absorption is saturable and non-saturable (7-10%)
3. Fully saturable in ileum but not jejunum (contrast with calcium)
4. Absorption in the colon significant
5. Vitamin D has no influence on magnesium absorption
Cation channel protein
(transient receptor
protein TRP)
Magnesium
Distal jejunum and ileum
TRPM6
ATP
Mg2+
Mg2+
ATP
ase
Mg2+
ADP
Mg2+ -bound to
phytate, fiber,
fatty acids
Enterocyte
Since TRPM6 operates by
diffusion without cotransporters, Mg2+
absorption efficiency
depends on the amount of
Mg2+ in the diet and within
the cell
Microminerals
3d metals: Fe, Zn, Cu
Microminerals
Fe2+, Cu2+, Mn2+, Zn2+
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.
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)
Zn(OH)2(s)
Zn2+(aq) + 2OH-(aq)
Solubility
Pulls equilibria
H+
Fe(OH)3 solubility
Zn(OH)2 solubility
1.0
2.0
3.0
4.0
5.0
pH
6.0
7.0
8.0
9.0
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
Mucins are complex polysaccharides secreted into the lumen
that 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
Correlation of spectra of Fe
with iron absorption
Importance of mucins
in making “insoluble”
iron available to
membrane
transporters
Rudzki et al, 1973
Conrad et al, 1991 as
cited in Powell et al, 1999
Stomach
(pyloric mucosa)
Laminated
mucous layer
Pyloric
mucosal
cells
Intestine (colon)
Mucous layer
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+ Pathway
Mobilferrin-integrin
Fe3+
Fe2+ Pathway
Divalent cation
transporter (DCT-1,
DCM-1,Nramp2)
Heme Pathway
Dctyb reductase
gastroferrin
Fe2+
Heme carrier protein
DCT1
integrin
Mobilferrin-Fe3+
Mobilferrin
Fe2+
Porphyrin ring
Ferroportin 1
Hephaestin
Cu
Cu
Cu
Iron Absorption (heme and non-heme)
Duodenal Mucosa
Duodenal Lumen
HemeProtein
Biliverdin
HFE
Heme
+
Polypeptides
Fe3+
Fe3+
Bilirubin
Heme
Ferritin
FR
Mucin
(gastroferrin)
DCT-1
B3 integrin
Fe2+
Fe3+
Bilirubin
CO
B2-microglobulin
Dcytb
reductase
Fe3+
Plasma
Fe3+
Fe2+
CO
Fe
2+
Ferroportin
paraferrin
Fe3+
Mobilferrin
(vesicles)
Hephaestin
Fe3+
Transferrin
Nramp2 (Natural resistance associated macrophage protein)
Nramp1
(no iron transport)
Nramp2
Nramp2
(DMT1/DCT1)
Transport Mn2+,Fe2+, Ni2+
DMT1 isoform 1
DMT1 isoform 2
Soluble mucins (gastroferrin)
Fe3+ reductase
FeR
DCT1
Mobilferrin
Integrin anchor
Secretions into the lumin (soluble mucins) retard hydrolysis of Cu, Fe and Zn
permitting binding to transporters and more efficient uptake.
Efficiency of transport is related to valance state with M+ > M2+ > M3+
Redox-active factors reduce Fe3+ to Fe2+
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.
Mobileferrin on the inner side of the apical membrane receives metal from
DCT1 and transfers it to cytosol.
Mobilferrin
HFE (human leukocyte antigen H)
Ferritin (or paraferritin) or Fe
2-microglobulin (for Zn)
HFE may be involved in stabilizing the above complexes to mobiltransferrin