gestion and Absorption of Minerals (Unifying principles that apply to all minerals)
Jan 14, 2016
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