Trace elements in clinical chemistry, major elements, minor elements, trace elements, iron, zinc, iodine, copper, manganese, fluoride, cobalt, molybdenum, selenium, boron, silicon, tin, cadmium, mercury, silver, titanium

1. Trace Elements
Dr. Umoh, Ofonmbuk

2. OUTLINE
• INTRODUCTION
• CLASSIFICATION
• INDIVIDUAL TRACE ELEMENTS
• FUNCTION
• SOURCES
• BODY REQUIREMENTS
• NUTRITIONAL DISORDER STATES
• MANAGEMENT OF DISORDER
• LABORATORY EVALUATION
• CONCLUSION
• REFERENCES
2

3. INTRODUCTION
The human body is composed of elements that can be
roughly divided into: Abundant, Major & Trace
elements.
• Abundant element: are those elements involved in
the formation of covalent bonds, and are important
constituents of tissues.
• They account for about 96% of the total body weight
e.g. hydrogen, oxygen, carbon, nitrogen.
• Their deficiencies can lead to energy – nutritional
disorders; and excesses causes obesity.
3

4. • The major elements, which often functions in the
ionic – cellular states, are those involved in the
maintenance of osmotic pressure and membrane
potentials e.g. sodium potassium, calcium etc. and
accounts for 3 – 4 % of the total body weight.
Deficiency or excesses of these elements often results in
water and electrolyte abnormalities.
• Trace elements of the human body are those elem-
ents accounting for about 0.02% of the total body
weight. Generally required in amounts less than 100mg
per day. They are inorganic analytes present at very low
concentration in the body – in nanograms to
micrograms per litre in body fluids, and micrograms to
milligrams per kg in tissues.
4

5. CLASSIFICATION
…of trace elements
The biological effects of deficiency state defines the
essentiality of a trace element, such that, an element
is considered essential when signs and symptoms
induced by its deficiency is reverse by an adequate
supply of the particular trace element in question.
• Essential trace elements: Iron, Zinc, Iodine, Copper,
Manganese, Fluoride, Cobalt, Chromium,
Molybdenum and Selenium.
• Probable essential: Lithium, Nickel, Boron, Silicon,
Tin, Cadmium & Vanadium.
• Non – essential: Lead Aluminum, Gold, Barium, lead,
Silver, Mercury, Strontium, Titanium, Zirconium. 5

6. • Reversal of clinical signs and symptoms by
supplementation with either a single trace
element, or micronutrient mixtures is often
used as clinical evidence of preexisting
deficiency.
• Indices used in this cases include:
• Growth velocities in children
• Regaining of lean body mass
• Rate of wound healing
• Resistance to infection
• Alteration in cognitive functions
6

7. • The wide range of biological activities and more
information on these elements has led to
classification beyond essentiality, into:
• Pharmacologically beneficial trace elements e.g.
fluoride used for protection against dental caries,
lithium salts used as mood stabilizers in bipolar
disorder and strontium for treatment of osteoporosis
etc.
• Nutritionally beneficial trace elements: for some
elements, continued suboptimal dietary intake – in
the presence of physiologic or metabolic stress – may
even have a detrimental effect for e.g. insufficient
Iodine and goiter.
7

8. • Note that:
• At low intakes of recognized essential trace elements,
deficient disease ensue. With increasing dietary
supply, a plateau region of optimal supply is reached,
and further intake could result in adverse toxic
effects.
• The concentration margin separating the beneficial
dietary intake from the toxic intake varies depending
on the elements, and may be very small, e.g. in
Lithium. Therefore, the Recommended Dietary
Allowance (RDA) is set at amount that is just
sufficient to prevent deficiency but not enough to
cause toxicity.
• Some metals such as Fe, Cu, Mo and Cr are stable in
more than one valence states and participates in
biologically important oxidation – reduction reactions.
8

9. • Specimens for the analysis of trace elements must be
collected with scrupulous attention to details such as
appropriate anticoagulant and specimen type (urine,
serum, plasma, or blood).
• Because of the low concentration in biologic
specimens and the ubiquitous presence in the
environment, extraordinary measures are required to
prevent contamination of the specimen. This includes
using special sampling and collection devices,
specially cleaned glassware, and water and reagents
of high purity.
9
General methods & instrumentation
for analyzing trace elements

10. …for analysis,
• No single technique is best for all purposes.
• The most commonly used instrumentations for
trace and toxic metal analysis has been
spectrophotometry: the atomic absorption (AA)
spectrometer, either with flame atomic absorption
(FAAS) or flameless (i.e. graphite furnace atomic
absorption spectroscopy [GFAAS]) atomization.
• Atomic emission spectrometry is also useful for
some elements, particularly if used in the form of
inductively coupled plasma atomic emission
spectroscopy (ICP-AES) for atomization and
excitation. 10

11. • Recently, inductively coupled plasma mass spectrometry
(ICP-MS) is becoming more widely used because of its
sensitivity, wide range of elements covered, and relative
freedom from interferences.
• Ion chromatography can be used for the determination
of copper, iron, and zinc in whole blood, serum, and
plasma and for the determination of zinc in urine.
• Gas chromatography–mass spectrometry (GC-MS) is
capable of determination of cadmium, chromium, cobalt,
copper, lead, and selenium in urine, copper in serum,
and lead in blood.
• The methods incorporating direct analysis of solid
samples, for instance, laser ablation ICP-MS (LA-ICPMS),
are gaining recognition for selected clinical applications.
11

12. 12
AA – Atomic Absorption spectrometer GFAA – Graphite Furnace Atomic Absorption spectroscopy
ICP-AES – Inductively Couped Plasma Atomic Emission Spectroscopy ICP-MS – “””” Mass Spectrometry

13. INDIVIDUAL TRACE
ELEMENTS
• Sources
• Body requirement
• Functions
• Nutritional disorder state
• Management of disorders
• Laboratory evaluation
13

14. Iron
• Considered to be the most essential trace element of
the body. Body content is about 3 – 4g, distributed as:
• Hemoglobin – (2000mg) 67%
• Storage forms: Ferritin, Hemosiderin – (1000mg)
27%
• Myoglobin – (130mg) 3.5%
• Labile pool – (8mg) 2.2% (peroxidases,
cytochrome, Kreb cycle enzymes)
• Other tissue iron – 0.2%
• Transport iron: apotransferrin & transferrin – 0.08%
14

15. • Dietary sources of iron includes: Leafy greens, whole
grains, beans, liver, spleen, mollusks (a type of sea
food)
• Daily requirement of iron: 0.5 – 2 mg normally, and 2
– 4mg/day in pregnancy.
• Daily excretion – about 0.9mg/day to 1.3mg/day
(during menstruation)
• Iron is essentially useful for delivery of oxygen to
cells; and as an agent in redox and electron transfer
reaction.
• About 10% of the dietary iron is absorbed, and this is
regulated by the hormone hepcidin.
15

16. • This takes place at the small intestine as Fe(2+). The
Fe(3+) forms must first be reduced to (2+) by ferric
reductase or vitamin C in the intestinal lumen before
absorption.
• In the intestinal mucosal cell, F(2+) can be bound by
ferritin for storage, or re-oxidized to (3+) and bound
to apotransferrin for transport throughout the body.
• After about 120days in circulation, RBCs are degraded
by the reticuloendothelial system, releasing Fe to the
circulation for reuse.
• Absorption and transport capacity can be increased in
conditions such as hypoxia & iron def. anemia. Each
menstrual cycle loses approx. 20 – 40 mg of iron.
16

17. • When iron is deficient, hemoglobin (Hb) cannot be
produced. Insufficient Hb leads to microcytic hypochromic
RBCs which are unable to deliver sufficient oxygen to the
tissues. This condition is known as iron deficiency anemia.
• This condition, depending on severity my be
asymptomatic, or may present with weakness, headache,
irritability and varying degree of fatigue/exercise
intolerance.
• Iron def. anemia can be treated with 3-6 mg of elemental
iron / kg/day. Available oral preparations include: ferrous
sulphate, ferrous fumarate, ferrous gluconate, succinate
iron and ferric aluminum citrate; where epigastric pain,
gastritis, metallic taste, nausea/vomiting, constipation or
diarrhea are known side effects.
• Parenteral preparations include iron dextran and iron
sorbitol citrate. Anaphylaxis, palpitations, headaches, fever
and injection site pains are the known adverse effects.17

18. • Conversely, excess iron in the body can be toxic
and life threatening. This can damage the intestinal
lining, or lead to shock and liver failure.
• Most common form of iron overload is
hemochromatosis, where there is iron accumulation
in tissues like the heart and liver. This can be due
to hereditary hemochromatosis – a primary Fe
overload, or secondary from excessive dietary, or
from medications as occurs in transfusion overload.
• Treatment of iron toxicity may include therapeutic
phlebotomy or administration of iron chelators such
as deferoxamine.
18

19. Laboratory evaluation of Iron
• Normal results for iron are: 65 to 175 mcg/dL for
men, and 50 to 170 mcg/dL for women.
• Disorders of iron metabolism are evaluated partly as
hematological or clinical chemistry assessment, by
packed cell volume, hemoglobin, red cell count and
indices, total iron content, total iron binding capacity,
percentage iron saturation, transferrin, and ferritin.
• Total Iron Content measures Fe bound to transferrin
using FAAS, or ID-MS. Specimen may be collected
either as serum or heparinized plasma.
• Total Iron Binding Capacity (TIBC) is the amount of
iron that could be bound by saturating transferrin and
other minor iron-binding proteins present in the
serum or plasma sample. This is measured using AAS
and ranges from 250 – 425 g/dL. 19

20. • Percentage iron saturation: also called transferrin
saturation, is the ratio of serum iron to TIBC. Normal is
20 – 50%, but it varies with age ad sex.
• Transferrin, which transport iron to various organs and
tissues, is increased in iron deficiency and decrease in
iron overload, hemochromatosis, chronic infections and
malignancies. Serum transferrin can be measured by
RIA, ELIA & Chemiluminescence. Normal values is 300
to 360 mcg/dL.
• Ferritin is an intracellular protein that stores and releases
iron in a controlled fashion, acting as a buffer against iron
deficiency and iron overload. It is decreased in iron
deficiency anemia, but increased in iron overload,
hemochromatosis, chronic infection, malignancy and viral
hepatitis. Ferritin is measured by chemiluminescence,
immunoradiometric (IRMA) assay and ELISA. 20

21. Zinc
• Zinc (Zn) is a bluish white lustrous metal (atomic No.
30, atomic wt 65.37) used in a production of alloys,
especially brass (with copper), in galvanizing, in
paints, in skin lotions, as treatment for Wilson’s
disease, and in many over-the-counter medications.
• It is an abundant trace element, essential for growth
& development.
• Zn is involved in the functioning of over 300 different
enzymes such as superoxide dismutase, alkaline
phosphatase and carboxypeptidase; and as cofactor
in DNA polymerase.
21

22. • Zinc is also important in reproduction, immune
regulation, collagen synthesis, wound healing,
convalescence, GIT modulation, preventing
dysmenorrhea.
• Body content - 2.5g: 60% in muscle, 30% in bone,
10% in body tissues and organs
• Daily requirement : 3 - 12 mg
• Diet rich in zinc : red meat, fish, sea food, pumpkin,
cashews, beans, dark chocolate
• Reference interval for zinc in serum is 80 – 120
mcg/dL, while zinc in urine of normal subjects is 0.2 –
1.3 mg/24hrs. Burn patients with acrodermatitis may
have zinc as low as 40 mcg/dL; these patients
respond quickly to zinc supplementation.
22

23. • Old age, pregnancy, lactation, and alcoholism are also
associated with poor zinc nutrition, and treatment of
leukemia, cirrhosis, hepatitis, sickle cell anemia as well
as malnutrition states have been observed to improve
better with addition of zinc.
• Zinc deficiency causes growth retardation, slow skeletal
maturation, testicular atrophy, hair loss, skin lesions
and reduced taste perception. Zinc deficiency is also
known to be associated with anorexia, cognitive and
motor impairment, diarrhea and pneumonia.
• Treatment is by zinc supplements 45 – 100 mg/day PO.
23

24. • Zinc is relatively non-toxic, though high doses of 1g or
repetitive doses of 100 mg/day for several months may
lead to disorders especially GIT symptoms.
• Exposure to ZnO fumes and dust may cause “zinc fume
fever.” The symptoms include chemically induced
pneumonia, severe pulmonary inflammation, fever,
hyperpnea, coughing, pains in legs and chest, and
vomiting.
• Zinc is measured by FAAS, ICP-AES, and ICP-MS. Low
urine zinc levels in the presence of low serum zinc
levels usually confirms zinc deficiency. Low serum zinc
in an apparently healthy (non-stressed and non-septic)
patient who has normal serum albumin levels can be
used as evidence of zinc deficiency, especially if urine
zinc levels are also low. 24

25. Copper
• Copper is an essential trace element found in
different oxidation states, Cu(1+) and Cu(2+), with
Cu(2+) being the most stable oxidation state.
• Richest dietary source : Organ meat, diet rich in
copper: red meat, shellfish, nuts, chocolate, seafood,
whole-grain foods. RDA – 1.3 - 1.5 mg/day
• Copper content in the normal human adult is 50 to
120mg. Copper is distributed through the body with
the highest concentrations found in liver, brain, heart,
and kidneys. Hepatic copper accounts for about 10%
of the total copper in the body. The amount of copper
absorbed from the intestine is 50% to 80% of
ingested copper.
25

26. • The average daily intake is approximately 10 mg or
more of copper. The exact mechanisms by which
copper is absorbed and transported by the intestine
are unknown, but an active transport mechanism at
low concentrations and passive diffusion at high
concentrations have been proposed.
• Copper is transported to the liver and bounds to
albumin, transcuprein, and low-molecular-weight
components in the portal system. In the liver, copper
is incorporated into ceruloplasmin (α2-globulin) for
distribution throughout the body.
• In a normal physiological state, 98% of copper
excretion is through the bile, with copper losses in
the urine and sweat comprising approximately 2% of
dietary intake
26

27. • Copper is a component, and cofactor of several
metalloenzymes, including ceruloplasmin, cytochrome
C oxidase, superoxide dismutase, tyrosinase,
metallothionine, dopamine hydroxylase, lysyl oxidase,
clotting factor V, and an unknown enzyme that cross-
links keratin in hair.
• Copper deficiency is observed in premature infants
and copper absorption is impaired in severe diffuse
diseases of small bowel, lymphosarcoma, and
scleroderma. Copper deficiency is related to
malnutrition, malabsorption, chronic diarrhea,
hyperalimentation, and prolonged feeding with low
copper, total-milk diets.
27

28. • Adequate intake – 34 mcg/kg. Deficient - <8.5 mcg/kg,
Toxic – 5mg/kg
• Signs of copper deficiency include: fragile hair, edema,
neutropenia and hypochromic anemia in the early stages,
osteoporosis and various bone and joint abnormalities that
reflect deficient copper-dependent cross-linking of bone
collagen and connective tissue, decreased pigmentation of
the skin and general pallor, and in the later stages,
possible neurologic abnormalities (hypotonia, apnea, and
psychomotor retardation).
• Subclinical copper depletion contributes to an increased
risk of coronary heart disease.
28

29. • An extreme form of copper deficiency is seen in
Menkes disease, an x-linked genetic disorder
with mutation in genes coding for the copper
transport protein ATP7A gene.
• Clinical signs include progressive mental
deterioration, coarse feces, disturbance of
muscle tone, seizures, and episodes of severe
hypothermia.
• Symptoms usually appear at the age of 3
months and death usually occurring by the age
of 5. This invariably fatal, progressive brain
disease is characterized by peculiar hair, called
kinky or steely, and retardation of growth.
29

30. • Copper toxicity has been associated with living near
copper-producing facilities, using water from copper
pipes or cooking with copper-lined vessels or
exposure to algaecides, herbicides, pyrotechnics,
ceramic glazes, electrical wiring, or welding supplies.
• Because of its redox potential, copper is an irritant to
epithelia and mucous membranes and can cause
hepatic and renal damage with hemolysis.
• Wilson’s disease is a genetically determined copper
accumulation disease that usually presents between
the ages of 6 and 40 years. Clinical findings include
neurologic disorders, liver dysfunction, and Kayser-
Fleischer rings (green-brown discoloration) in the
cornea caused by copper deposition.
30

31. • Early diagnosis of Wilson’s disease is important
because complications can be effectively prevented
and in some cases the disease can be halted with the
use of zinc acetate or chelation therapy.
• TREATMENT : Life long.
• Avoidance of high Copper diet
• In early stages Zn may be effective as it competes
with Cu for absorption.
• REFERENCE INTERVALS OF COPPER :
• Copper in serum : 700–1500 g/L.
• Copper in serum (pregnancy at term) : 118–320 g/L
• Copper in urine : 15–60 g/24 hours.
31

32. • Serum ceruloplasmin levels and the direct
measurement of free copper are key
diagnostic steps in the diagnosis of Wilson’s
disease.
• Copper is measured by flame AAS, ICP-MS,
ICP-AES, and ASV.
• Serum copper and urine copper are used to
monitor for nutritional adequacy and during
management of copper toxicity.
• Direct measurement of free copper and
ceruloplasmin in serum is used to screen for
Wilson’s disease.
32

33. Iodine
• More than half of the iodine is found in thyroid
gland. Iodine is an essential component for
production of thyroid hormones, including
thyroxine.
• Most of the iodine in the diet comes from sources
such as: cheese, cows milk, eggs, iodized table salt,
saltwater fish, shellfish, soy milk.
• The RDA is about 150 µg per day in adults. 220 µg
iodine per day for pregnant women and 290 µg
iodine per day for breastfeeding women.
• Consuming diets high in goitrogens such as
cabbage, cassava and millet, limits the
bioavailability of Iodine. 33

34. • The thyroid gland has the ability to accumulate
iodide (I−) against a steep electrochemical gradient
via a Na+/iodide symporter.
• Thus, the iodide concentration in the cytoplasm of
thyroid follicular cells, as well as the lumen of
thyroid follicles can be many folds higher than that
in plasma.
• Iodine deficiency is defined as a median urinary
iodine concentration less than 100µg/L in a
nonpregnant population, or <150 µg/L in a
population of pregnant women.
34

35. • Almost all the symptoms of iodine disorders are
related to its effect on the thyroid: Without adequate
iodine, the thyroid progressively enlarges as it tries to
keep up with demand for thyroid hormone production.
• Worldwide, iodine deficiency is the most common
cause of thyroid enlargement and goiter.
• Severe iodine deficiency in the mother has been
associated with miscarriages, stillbirth, preterm
delivery, and congenital abnormalities in their babies.
• Children of mothers with severe iodine deficiency
during pregnancy can have intellectual disabilities and
problems with growth, hearing, and speech. In the
most severe form, an underactive thyroid can result in
cretinism (a syndrome characterized by permanent
brain damage, deaf mutism, spasticity, and short
stature), 35

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39. • Iodine deficiency prophylaxis : iodized salt, iodized
oil, iodized water, iodine tablets or drops, certain
ointment, solution, mouth gargles.
• For determining serum inorganic iodide (SII) the
HPLC assay is the method of choice, because
contaminations from the protein bound iodine
fraction do not interfere with the detection process.
• The clinical relevance of the measurement of SII is
limited, but allows the calculation of the absolute
iodine uptake which has great value in
pathophysiologic studies
39

40. Chromium
• Chromium exists in two main valency states:
trivalent and hexavalent.
• Chromium(VI) is better absorbed and more
toxic than chromium(III) and has also been
listed as a carcinogen implicated in lung
cancer.
• Regulates plasma lipoprotein concentration and
reduces cholesterol and triglycerides.
• Found in – grains, cereals, fruits, processed
meat.
• RDA : 20 – 35 mcg/ day
40

41. • Reference Intervals for Chromium :
• In whole blood: 0.7–28.0 g/L
• In serum: 0.05–0.5 g/L
• In urine: 0.1–2.0 g/24 hr
• In RBCs: 20–36 g/L
• DEFICIENCY :
• Chromium deficiency is characterized by glucose
intolerance, glycosuria, hypercholesterolemia,
decreased longevity, decreased sperm counts, and
impaired fertility
41

42. • TOXICITY :
• Lung Carcinoma, Bronchogenic Carcinoma in
stainless steel workers, Dermatitis, skin ulcers
• Chromium may be determined by GFAAS, NAA, or
ICP-MS. Plasma, serum, and urine do not indicate
the total body status of the individual, whereas
urine levels may be useful for metabolic studies.
42

43. MANGANESE
• Shown to be essential for normal growth
and development
• Role: Manganese superoxide dismutase,
Arginase, Glutamate synthase & Pyruvate
carboxylase
• RDA – 2-11 mg/day
• Dietary sources: meat, fish, poultry, dry
fruits and nuts
43

44. • DEFICIENCY
• Experimental animals - ↓growth, ↓fertility,
ataxia, skeletal deformities, abnormal fat
and CHO metabolism
• TOXICITY :
• Neurotoxic- extrapyramidal parts affected
Headache
• Hepatic dysfunction
44

45. Molybdenum
• Molybdenum (Mo) is a hard silvery white
metal.
• Act as a catalyst for enzymes and helps
facilitate breakdown of certain amino acids,
corrosion inhibitors, flame retardants, smoke
repressants, and molybdenum blue pigments.
• Molybdenum in human tooth enamel may have
a role in lowering the risk of tooth decay.
• RDA : 45mg/day.
• Pregnancy and Lactation - 50mg/day
45

46. • REFERENCE INTERVALS FOR MOLYBDENUM
• Molybdenum in whole blood: 60 g/L
• Molybdenum in serum: 0.1–3.0 g/L
• Molybdenum in red cells: 18 g/L
• Molybdenum in urine: 8–34 g/L
46

47. • DEFICIENCY :
• Molybdenum cofactor deficiency is a recessively
inherited error of metabolism.
• Symptoms : include seizures, anterior lens
dislocation, decreased brain weight, and usually
death prior to age 1 year.
• TOXICITY :
• High dietary and occupational exposures to
molybdenum have been linked to elevated uric acid
in blood and an increased incidence of gout.
47

48. Selenium
• Less abundant trace element
• Recommended intake for adults 50-200 μg/day
• Functions in human body:
• Selenium in Glutathione peroxidase (GTH-Px) -
important role in protecting the body from
oxidative damage; in immune system function,
and also plays a crucial role in the control of
oxygen metabolism.
48

49. • Protects phagocytes from destruction
• GTH-Px protects eye lens tissues and neurons
from damage
• DEFICIENCY
• Selenium deficiency occurs due to: Hemolytic
anemia, Clansman's thrombasthenia (platelet
disorder), Gastrointestinal cancer, Malnutrition
• Selenium deficiency has been associated with
cardiomyopathy, skeletal muscle weakness, and
osteoarthritis.
49

50. • A significant negative correlation was observed
between selenium intakes and cancer of the
large intestine, rectum, prostate, breast,
ovary, and lungs and leukemia.
• Keshan disease : An endemic cardiomyopathy
that affects mostly children and women in
childbearing age in certain areas in China, has
been associated with selenium deficiency.
50

51. Boron
• Boron is a vital trace mineral that is required for the normal
growth and health of the body.
• Apples, oranges, red grapes, kiwis, dates, as well as certain
vegetables, avocado, soybeans and nuts are rich sources of
boron
51

52. • Health benefits of Boron:
• Prevents arthritis
• Used for bodybuilding
• Estrogen Production: Boron can improve the
production of estrogen in menopausal
women
• Embryonic development: Boron appears to be
essential for reproduction and the
development of the fetus
• Proper cell membrane functions
• Lowers plasma lipid levels 52

53. Summary & Conclusion
• In clinical chemistry, an trace element is a dietary
element that is needed in very minute quantities for the
proper growth, development, and physiology of the
organism. The essential trace elements are those that
are required to perform vital metabolic activities in the
body.
• Examples of essential trace elements in animals include
Fe (hemoglobin), Cu (respiratory pigments), Co
(Vitamin B12), Mn and Zn (enzymes). Some examples
within the human body are cobalt, copper, fluorine,
iodine, iron, manganese and zinc. Although they are
essential, they become toxic at high concentrations.
Elements such as Ag, As, Cd, Cr, Hg, Ni, Pb, and Sn
have no known biological function, with toxic effects
even at low concentration. 53

54. 54
References
• Bishops Clinical Chemistry – Principles, Techniques, Correlations, 7th Ed.
• Martin A. Crook; Clinical Chemistry and Metabolic Medicine, 5th Edition
• Tietz (Full) Textbook of Clinical Chemistry and Molecular Diagnostics, 5E
• Harrison’s Principles of Internal Medicine, 18th Ed, 2012
• Lichtenstein AH, Russell RM: Essential nutrients: Food or supplements?
Where should the emphasis be? JAMA 294:351, 2005[PMID: 16030280]
• German Nutrition Society: Reference values ​​for nutrient intake (DACH).
1st edition. Frankfurt am Main: Umschau / Braus, 2000. pp 216 – 221.

55. Thank you for
listening

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