BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 1
Technische Universität München
Assessing bioavailability of
essential trace minerals
in animal nutrition
Wilhelm Windisch
Hans-Eisenmann-Zentrum
Center of Life and Food SciencesWeihenstephan
Technische Universität München, Germany
Chair of Animal Nutrition
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 2
Technische Universität München
In vitro studies cannot fully
cover bioavailability
Bioavailability of essential trace minerals:restrictions resulting from general definition
…the maximum possible yield of a nutrient
that the body may extract from the ingested food and use for its metabolic functions…
(Kirchgessner et al. 1993)
bio – availability
metabolic(re)actions
dietaryproperties
Bioavailability= capability
of metabolic
use at the absence of
homeostatic regulation
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 3
Technische Universität München
0
200
400
600
800
1000
1200
0 200 400 600 800 1000 1200
Zn intake (µg/day)
(µg Zn/day
Zn intake
Zn retention
Zn in feces
(Windisch and Kirchgessner 1995)
require-ment
Zn exceeding requirement
is rejected from absorption
E.g. homeostatic regulation of Zn metabolism:Precise control of Zn uptake from intestinal tract
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 4
Technische Universität München
Metabolic use of dietary trace mineralsin relation to homeostatic counter-regulation
homeostatically restricted
use of dietary TM available in excess to
requirement
homeostatic regulation
at sufficient supply
maximum possible useof dietary TM (capability)
deficient
supply
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 5
Technische Universität München
13
14
15
16
17
18
0 100 200 300 400 500 600
Zn content of feed (ppm)
Zn content (ppm)
homeostatic regulationat sufficient Zn supply
Concentration
of Zn in the egg
e.g. Zn homeostasis: Zn concentration in eggs
(Paulicks and Kirchgessner 1994)
onset of homeostatic regulation
homeostasis is locked at maximum use,
dose-response relationship
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 6
Technische Universität München
0
2
4
6
8
10
0 25 50 75 100 125 150
dietary Zn (mg/kg DM)
Zn in m
ilk (µg/g)
Range of homeostatic regulation
Zn content in cow milk
Defi-
ciency
Example Zn homeostasis:Zn steady state in products (e.g. milk)
(Schwarz and Kirchgessner 1975)
onset of homeostatic regulation
homeostasis is locked at maximum use,
dose-response relationship
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 7
Technische Universität München
e.g. Se homeostasis: whole body Se retention
deficiency excesshomeostasis
(Kirchgessner et al.1997)
3000 p
pb
300 p
pb
600 p
pb
1000 p
pb
200 p
pb
150 p
pb
70 p
pb
100 p
pb
40 p
pb
450 p
pb
Homeostasis
is locked atmaximum use,
dose-response
relationship
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 8
Technische Universität München
e.g. Se homeostasis: urinary Se excretion
(Kirchgessner et al.1997)
deficiency excesshomeostasis
onset of
homeostatic
regulation
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 9
Technische Universität München
Inorganic vs. organic Se compounds
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 10
Technische Universität München
General principle to assess bioavailability of trace minerals
TM intake
Re
sp
on
se
para
me
ter
de
ficie
nt
su
ffic
ien
t
range of dietary
supply to assess bioavailability
(deficiency)
Dietary requirement at high bioavailability
(onset of homeostatic counter-regulation)
Low BA
High BA
Different slope = different bioavailability
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 11
Technische Universität München
In vitro studies cannot fully
cover bioavailability
Bioavailability of essential trace minerals:restrictions resulting from general definition
…the maximum possible yield of a nutrient
that the body may extract from the ingested food and use for its metabolic functions…
(Kirchgessner et al. 1993)
bio – availability
metabolic(re)actions
dietaryproperties
Physiological conditions of metabolism,
sensitive response parameter
Bioavailability= capability
of metabolic
use at the absence of
homeostatic regulation
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 12
Technische Universität München
Metabolic use of dietary trace mineralsin relation to homeostatic counter-regulation
homeostaticallyrestricted use
homeostatic regulation
at sufficient supply
max. possible use
deficient
supply
Dietary excess overwhelms
homeostasis(accumulation)
excess � toxicity
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 13
Technische Universität München
Short term oral excess demonstrates ability to overwhelm homeostatic counter-regulation (AUC-method)
Zn glycine Zn sulfate Zn lactate Zn oxide
placebo level
(Single oral Zn load in horses: Wichert et al. 2001)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 14
Technische Universität München
In vitro studies cannot fully
cover bioavailability
Bioavailability of essential trace minerals:restrictions resulting from general definition
…the maximum possible yield of a nutrient
that the body may extract from the ingested food and use for its metabolic functions…
(Kirchgessner et al. 1993)
bio – availability
metabolic(re)actions
dietaryproperties
Physiological conditions of metabolism,
sensitive response parameter
Interactions withother dietary
components
Bioavailability= capability
of metabolic
use at the absence of
homeostatic regulation
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 15
Technische Universität München
0 2 4 6 8 10 12
Corn
wheat
barley
soya
extracts
rape seed
extracts
P content (g/kg DM)
phytate P
digestible P
Phytic acid (phytate)
Inositol 1,2,3,4,5,6-Hexakis-dihydrogenphosphat
(Lantzsch 1990)
Phytic acid is a strong chelator to trace minerals
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 16
Technische Universität München
(Windisch and Kirchgessner 1999)
Dietary phytate may massively reducemaximum possible Zn absorption measured at Zn deficiency
Almost 100% absorption from diets without chelators
Range of phytate
content in common
diets to pigs/poultry
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 17
Technische Universität München
-5
0
5
10
15
20
Control Zn deficiency Zn deficiency plus
added phytase
apparetn Zn absorption (%)
-0,25
0,00
0,25
0,50
0,75
1,00
Zn in blood plasm
a (ppm)
Zn absorption
Zn in blood plasma
(750 FTU/kg(140 ppm Zn) (30 ppm Zn)
Added phytase may significantly improve Zn bioavailability(e.g. from inorganic sources, Zn sulfate)
(Ettle et al. 2006)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 18
Technische Universität München
Se retention from organic Se depends onsupply status with methionine (growing rat model)
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
Added methionine (%)
tissue Se (ppb)
(Butler et al. 1989)
Liver
Whole blood
Muscle
Basal diet: marginal in Met, 800 ppb dietary Se as SeMet
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 19
Technische Universität München
Assessing bioavailability of essential trace mineralsin animal nutrition
In vitro methods do not fully cover bioavailability (BA).
BA = capability of metabolic trace mineral use
It is not fully realized at normal feeding conditions
Assessment of BA at deficient trace mineral supply(no interference with homeostasis).
BA cannot be assessed independent from dietary composition.
Example to Zinc
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 20
Technische Universität München
Example: Quantifying Zn bioavailability with a radiotracer study
Bioavailability metabolic utilization of
absorbed dietary Zn
…for tissue retention,endogenous faecal
and renal excretion,surface losses, …
true absorption
of dietary Zn
= influx of Zn fromdiet into the
inside of theorganism
= x
measured in a radiotracer study
at Zn deficiencyusing a purified diet added with Na12phytate (8g/kg)
(Schlegel and Windisch 2006)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 21
Technische Universität München
Example: Quantifying Zn bioavailability in a radiotracer study(Schlegel and Windisch 2006)
positive controlsulfate
(52µg/g)
sufficient Zn
1.35 a
516
159 a
48 a
4 a
107 a
Test group
Zn glycinate,(12µg/g)
deficient Zn
0.76 b
10956 b
18 b
2 b
35 b
50.8 a
95.748.6 a
negative control
sulfate(12µg/g)
deficient Zn
0.71 b
10848 b
18 b
3 b
27 b
44.2 b
94.741.8 b
Treatment group:
Added dietary Zn
Zn status
Blood plasma Zn (µg/ml)
Zn flux (µg/day)
intaketruly absorbed from diet
endogenous faecal excretionurine
retention
Max. absorption (%)
Metabolic utilization (%)Bioavailabiltiy (%)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 22
Technische Universität München
Experimental model to assess Zn bioavailabilityin practical pig feeding
Diet?
Zn supply before the onset of
study: depletion or adequate?
“Worst case” diet (corn & soyban extracts):
rich in phytate, low in native Zn, no phytase
activity (pelleted with steam). Graded levels of added Zn (sulfate) from deficient to sufficient
supply
(Brugger et al. 2012)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 23
Technische Universität München
Mobilization and refilling of mobilized bone Znis highly regulated by homeostasis (rat model)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 24
Technische Universität München
Experimental model to assess Zn bioavailabilityin practical pig feeding
Diet?
Zn supply before the onset of
study: depletion or adequate?
Duration of Zn deficiency?(no Zn deficiency symptoms)
“Worst case” diet (corn & soybean extracts):
rich in phytate, low in native Zn, no phytase
activity (pelleted with steam). Graded levels of added Zn (sulfate) from deficient to sufficient
supply
Adequate Zn supply before the onset of study
(Brugger et al. 2012)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 25
Technische Universität München
Symptoms of Zn deficiency in piglets fed asoya-corn-based diet without Zn supplementation
(Windisch et al. 2003)
Futterverzehr von Ferkeln unter Zn-Mangel
0
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700
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900
1000
0 2 4 6 8 10 12 14 16 18 20 22 24
Tag des Zn-Mangels
Futterverzehr (g/Tag)
normal
Zn-Mangel
Futterverzehr von Ferkeln unter Zn-Mangel
0
100
200
300
400
500
600
700
800
900
1000
0 2 4 6 8 10 12 14 16 18 20 22 24
Tag des Zn-Mangels
Futterverzehr (g/Tag)
normal
Zn-Mangel
Futterverzehr von Ferkeln unter Zn-Mangel
0
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500
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1000
0 2 4 6 8 10 12 14 16 18 20 22 24
Tag des Zn-Mangels
Futterverzehr (g/Tag)
Futterverzehr von Ferkeln unter Zn-Mangel
0
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1000
0 2 4 6 8 10 12 14 16 18 20 22 24
Tag des Zn-Mangels
Futterverzehr (g/Tag)
normal
Zn-Mangel
Futterverzehr von Ferkeln unter Zn-Mangel
0
100
200
300
400
500
600
700
800
900
1000
0 2 4 6 8 10 12 14 16 18 20 22 24
Tag des Zn-Mangels
Futterverzehr (g/Tag)
Futterverzehr von Ferkeln unter Zn-Mangel
0
100
200
300
400
500
600
700
800
900
1000
0 2 4 6 8 10 12 14 16 18 20 22 24
Tag des Zn-Mangels
Futterverzehr (g/Tag)
normal
Zn-Mangel
Feed intake of piglets at „native“ Zn supply
day of feeding
(g/d
ay)
control
feed with native Zn
Maximum
duration of physiologi-
cally tolerable Zn deficiency
Final exhaustion of mobilizable Zn stores
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 26
Technische Universität München
Experimental model to assess Zn bioavailabilityin practical pig feeding
Diet?
Zn supply before the onset of
study: depletion or adequate?
Duration of Zn deficiency?(no Zn deficiency symptoms)
“Worst case” diet (corn & soybean extracts):
rich in phytate, low in native Zn, no phytase
activity (pelleted with steam). Graded levels of added Zn (sulfate) from deficient to sufficient
supply
Adequate Zn supply before the onset of study
Maximum 8 days
Response parameter? • Apparently absorbed dietary Zn (mg/day)• Blood plasma: total Zn, AP activity
• Bone Zn• mRNA of metallothioneine in intestinal tissues
(Brugger et al. 2012)
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 27
Technische Universität München
Reaction of apparently absorbed dietary Zn indicatesabsence/presence of homeostatic counter-regulation
Zn deficiencysufficient Zn supply
(homeostasis)
break point at 59mg/kg
(Brugger et al. 2012)
theoretical max. slope of 1.0 (100% BA)
Relevant range of dietary Zn to compare Zn sources of unknown bioavailability.
measured slope = 0.23
native � added
BfR Berlin, Jan 16th-17th, 2013 W. Windisch: Assessing bioavailability of essential trace minerals in animal nutrition 28
Technische Universität München
Assessing bioavailability of essential trace mineralsin animal nutrition
In vitro methods do not fully cover bioavailability (BA).
BA = capability of metabolic trace mineral use. It is not fully realized at normal feeding conditions.
Assessment of BA at deficient trace mineral supply (no interferencewith homeostasis) and absence of deficiency disorders
BA cannot be assessed independent from dietary composition.
Comparison of dietary trace mineral sources for BA should be done on
base of a well defined standard “worst case” diet.