TRACE METALS 2023.pdfjesmiemma89@gmail.com.un

TRACE METALS
DR MRS SANDRA A N A CRABBE
MGCP, MBChB

OBJECTIVES
• Reminder of what trace elements are and their function
• Factors to consider in laboratory assessment of trace elements
• Study of some specific trace elements

Introduction
• The maintenance of normal health requires provision in the diet of adequate protein,
energy substrates, vitamins and also of various inorganic salts and trace elements
• Trace metals are inorganic micronutrients that are present in very low
concentrations in body fluids and tissues.
• Those present in body fluids (pg/dl) and in tissue (mg/kg) are, widely referred to as
"trace elements" and those found at ng/dl or ug/kg as the "ultratrace elements”
• Their dietary requirements are in μg to mg/day.
• They are required for the proper functioning of many enzymes and other proteins.
• Their deficiency can lead to specific signs and symptoms. Sometimes the deficiency is not
dietary but due to transport or recycling defects.
• In humans, metals are obtained exogenously and require intestinal absorption and
transport to the appropriate intra-cellular compartment for function.

Introduction
• An element is considered essential when the signs and symptoms induced by a
deficient diet are uniquely reversed by an adequate supply of the particular trace
element
• For iron, iodine, cobalt (as cobalamins), selenium, copper, and zinc, there are
clinical examples of reversible deficiency disease. Enough is known about the
biochemical functions of these elements to explain their importance in human
nutrition.
• For others, such as manganese, chromium, molybdenum, and vanadium, their
importance remains to be fully accepted in clinical practice
• Other elements, such as bromine, fluorine, cadmium, lead, strontium, lithium and
tin, have been claimed by at least one investigator to be essential for one or more
animal species, as demonstrated by dietary deprivation studies
• It is important to note that several of these metals are toxic at higher
concentrations

Some trace elements
Element Function
Chromium Component of chromodulin (potentiates insulin)
Cobalt Component of vitamin B12
Copper Cofactor of cytochrome oxidase
Fluorine Present in bone and teeth. Function?
Iodine Component of thyroid hormones
Iron Component of haem pigment
Manganese Cofactor for several enzymes
Molybdenum Cofactor for xanthine oxidase
Selenium Cofactor for glutathione peroxidase
Silicon Present in cartilage. Function?
Zinc Cofactor for many enzymes

Some form of classification
• Pharmacologically beneficial: Trace elements in this category include
fluoride used for protection against dental caries, lithium salts used in the
treatment of manic depression, and strontium ranelate for the treatment
of osteoporosis. Dosages required for a beneficial pharmacologic effect
greatly exceed the amounts of these elements normally found in food.
• Nutritionally beneficial or possibly essential: For some trace elements,
continued suboptimal dietary intake (in the presence of physiologic,
nutritional, or other metabolic stress) may eventually have a detrimental
effect for which dietary supplementation may have a ‘health restorative’
effect. Such effects are most clearly demonstrated in experimental animals.
Examples include the effects of boron in the presence of vitamin D
depletion and the need for increased vanadium with an experimentally
induced deficient or excessive supply of dietary iodine.

General knowledge on trace elements
• Various reviews have investigated epidemiologic evidence of the
involvement of trace elements, especially copper, zinc,and selenium,
in influencing cancer risk; it is clear that evidence is relatively weak,
and the need for more detailed study remains.
• A systematic review of most of the trace and toxic elements
measured in plasma or whole blood in haemodialysis patients
suggested that zinc and selenium were consistently below normal
values, whereas copper and chromium were within reference limits.
The authors recommended more careful monitoring of this group of
patients, for whom intake is easily compromised

Consequences of inadequate mineral or trace
element intake
• Optimal tissue function with body stores (if any) replete
• Mobilization of stores (if any)
• Initial depletion : Compensation (if possible) by increased absorption from gut,
reduced renal excretion, reduced growth velocity (Zn)
• Intracellular content reduced
• Impaired biochemical function : Reduced intracellular enzyme activity (metabolic
effects, antioxidant systems), Gene expression/regulation
• Non specific functional effects : Short term (cognitive effects, fatigue,
immunologic function); Long term (Free radical damage to DNA/cell membranes)
• Clinical disease : Typical for each trace element (complicated if multiple)
deficiencies
• Death

• At low intakes of recognized essential trace elements, deficiency
disease may be seen; with increasing dietary supply, a plateau region
of optimal supply is reached. Still higher intakes will result in adverse
toxic effects
• Reversal of clinical signs and symptoms by supplementation with a
single trace element or with micronutrient mixtures has been used as
indirect evidence of a preexisting deficiency. Growth velocities in
children, regain of lean body mass, rate of wound healing, resistance
to infection, and alterations in cognitive function can be assessed.
However, many confounding factors, in particular the presence of
disease or of other nutritional deficits, can affect the interpretation of
changes in these indices.

• Requirements for most micro- and macronutrients that are required to
maintain health have been characterized and made available in reports
from the Institute of Medicine (IOM) of The National Academies.
• The effects of disease may increase physiologic demands for such nutrients
e.g. , hypermetabolism, as a result of trauma or infection, increases the
requirements for protein, energy, and micronutrients. Increased losses of
nutrients from the gut, kidney, and skin, or through dialysis, may also
increase overall nutrient demands.
• Reductions in metalloenzyme activity induced by the deficiency may be
partially or wholly restored by effective treatment. Reversal of
haematologic and immune function laboratory abnormalities can also be
addressed, as can hormonal changes induced by the deficiency

Chemistry and Metal Interactions
• Trace elements interact with available ligands, mainly the electron donors
(e.g., nitrogen, sulfur, and oxygen) to form a wide variety of chemical
complexes or species.
• Some metals such as Fe, Cu, Mo, and Cr are stable in more than one
valence state and participate in biologically important oxidation-reduction
reactions.
• The transition metals (e.g., Fe, Cu, and Co), with an incompletely filled 3d
orbital, coordinate with a large number of groups to form stable
complexes.
• Zn lies at the end of the first transition series in the periodic table, Zn2+,
with a complete 3d electron shell, is a particularly stable ion with unique
biological functions.

Chemistry and metal interactions
• Bioavailability from foods and drinks depends mainly on solubility and the
presence of other dietary components which either promote or inhibit
absorption. The formation of soluble complexes with citric and other organic
acids is also important.
• Absorption of these organo-metallic complexes will be facilitated through organic
acid transporters. Iron, in the form of heme-Fe derived from meat products, is
more readily absorbed than inorganic Fe. However, other dietary components can
inhibit absorption by reducing solubility. The molybdate ion can form insoluble
Cu-molybdate complexes in the intestine, limiting absorption. Phytate and fiber
form insoluble complexes with Zn with well described defeciency resulting.
• Some interelement interactions also occurs. It is known that increasing dietary
intake of Zn reduces Fe uptake and vice versa. The species of the element and its
solubility are important because different salts of Zn can produce varying effects

Chemistry and metal interactions
• An excessive intake of Zn induces synthesis of intestinal metallothionein
that traps the metal in the cell which is eventually shed into the lumen. As
metallothein also strongly binds Cu, this is also lost and the body load can
become severely depleted.
• Synergistic interactions occur in other tissues and can have important
biological and clinical consequences. E.g., the interaction between
selenium and iodine has been investigated. The deiodinase enzymes that
remove I from T4 to produce the biologically active T3 are selenoproteins.
Also, the selenoprotein glutathione peroxidase is active in thyroid gland to
decrease excess hydrogen peroxide formation. Selenium is important,
therefore in thyroid hormone metabolism. In certain areas of the world,
combined Se and I defeciency occurs and provision of Se may be necessary
to correct hypothyroidism, but this also may precipitate its onset.

Biochemistry/homeostasis of trace elements
• Most aspects of intermediary metabolism require essential trace elements in the
form of metalloenzymes that have a number of catalytic properties
• Specific metalloproteins are required for the transport and safe storage of very
reactive metal ions such as Fe3+ or Cu2+ such as metallothionein (Cu, Zn),
transferrin, ferritin and haemosiderin (Fe) and caeruloplasmin (Cu)
• Supply of essential trace elements to tissue cells are under homeostatic control
• The controls include: intestinal absorption, specific transport systems in
peripheral blood, uptake and storage mechanisms in tissue and control of
excretion.
• The principal excretory route for some important trace metals (Cu, Zn) is in faeces
by regulation of initial absorption and by resecretion into the intestinal tract and
bile and other intestinal fluids. In general, the more the intake, the greater the
overall excretion associated with increasing overall element retention

Biochemistry/homeostasis
• For the halides (iodide and fluoride), excess intake is excreted primarily in urine
• For others (Se, B, Mo, Cr) urinary output is also important
• Loss of trace elements via other routes e.g. hair/nails, skin cell desquamation and
sweat is generally minor
• Menstrual loss of Fe and seminal fluid loss of Zn could be important in specialized
studies
• Overt symptomatic deficiency disease may result from combinations of poor
dietary supply, intestinal malabsorption (due to antagonistic effects of other trace
elements and blockage of uptake by substances like phytates) and increased
excretory losses (disease, injury, infection).
• Catabolic responses to injury, infection and malignant disease can lead to an
increase in losses in faeces and urine whilst burn injury causes extensive loss in
exudates through the damaged skin

Inborn Errors
• Genetic defects in the metabolism of trace elements are rare, but,
important because of the information they provide as to homeostatic
control mechanisms.
• This information has led to the development of effective therapeutic
strategies.
• The most commonly investigated disorders are those affecting Fe
(haemochromatosis), Cu (Wilson disease and Menkes syndrome), Zn
(acrodermatitis enteropathica [AE]), and Mo (molybdenum cofactor
disease).

Clinical Applications
• Nutritional support : There are Recommended Daily Allowance (RDA) for healthy
individuals. Increased exposure, with a risk of toxicity, can occur where there is
contamination of food, drinking water, or other beverages. Trace element
support to patients receiving enhanced enteral or parenteral nutrition is common
practice (due to feeding difficulties). Monitoring trace elements in treatment of
some of the genetic disorders is very important. Important in monitoring patients
post-bariatric surgery.
• Prostheses and Implants : Metallic components of orthopaedic devices and other
implants represent a source of internal exposure as surface reactions such as
corrosion and wear cause release into surrounding tissue and into circulation.
Investigations have been directed toward two primary questions; is release of
metal associated with functional failure of the implant and is there any toxicity
associated with the release of these metals?
• Pharmacologic uses : E.g. lithium in managing depression

Laboratory assessment of trace element
status
• As understanding of underlying biochemical intracellular mechanisms increases for a
particular trace element, the determination of active species becomes of increased
importance.
• Direct determination of iodine has been largely replaced by assay of thyroid hormones
and their control and feedback
• Cobalamin (vitamin B12) is measured in body fluids by immunoassay rather than
determination of cobalt which is nonspecific
• Work is ongoing on other trace elements in similar regard because metalloenzymes and
protein species are proposed as indices of Fe, Zn, Cu and Se status
• There are biological parameters for assessing iron status, whilst those for Zn and Cu are
not routinely used (zinc-induced metallothionein monocyte mRNA relates to low Zn
intake)
• With regards to the ‘ultra trace elements’ information is still being sought about the
critical molecular species responsible for their biological actions and therefore direct
determination of total concentration in body fluids are used. Require specialist lab facility

Lab assessment of status
• Estimation of dietary intake has been done by direct dietary analysis and
dietary history documentation for individuals and populations
• Estimation of positive or negative balance has been done by direct
measurement of total dietary intake over several days together with
measurement of all outputs in urine, faeces or any other route (challenge is
intrinsic difficulty with complete collection of sample with minimal
contamination)
• Estimation of net intake can be done by the use of exogenous and
endogenous labeling of representative diets with radioisotopes or stable
isotopes of the trace element under investigation. This can give great
insight into the bioavalability of nutrients and efficiency of uptake.

General considerations in analyses of trace
elements
• Specimen Requirements:
a) Direct determination can be done in various specimen including whole blood, blood plasma or
serum, leucocytes, urine, saliva, CSF, breast milk and sweat. Tissue samples may be obtained
by needle biopsy (liver, bone) or following autopsy. Hair and nail samples are useful non-
invasive options to assess toxic metal exposure but for essential elements , may be of value on
a group basis during studies of severely depleted populations but limited in value for
investigating individual hospital patients. Problems of external contamination from
environmental pollution, cosmetics, shampoos etc are very real.
b) Plasma protein concentration of the relevant carrier proteins provide useful additional
information (transferrin, albumin, caeruloplasmin and selenoprotein P) when using blood.
c) Direct measurement of intracellular concentration in nucleated cells (leucocytes and platelets).
Challenge is with contamination during the process of separation of different white cells and
platelets in whole blood
d) Problems from prolonged storage of samples and repeated freezing and thawing not
encouraged

General considerations………
• Preanalytical factors:
a) Guidelines giving details of sample collection procedures and procedures
for limiting contamination should be made available
b) Age, sex, ethnic origin, time of sample in relation to food intake, time of
day and year, history of medication, tobacco usage etc should be
recorded when reference intervals are established from healthy controls
c) In-patients with infection, post-trauma, post-surgery, systemic
inflammatory response can affect essential trace element concentrations
independent of their nutritional status. Typical example is in relation to
acute phase reactants (APR) which can cause an increase in permeability
of capillary allowing transfer of some plasma carrier proteins and their
trace metals into the interstitium or induction of hepatic synthesis of the
APRs and therefore elevated with any metals they carry.

General considerations……..
• Collection equipment: contamination is the major issue
a) For blood plasma, plastic tubes with lithium heparin as an anticoagulant are suitable for most
analyses.
b) For blood serum, plain glass containers are used (avoid containers with gel clotting agents)
c) For the ultratrace metals (Mn, Cr), special arrangements have to be made to collect blood via
plastic cannulae or silanized steel needles, and then the sample is placed into acid-washed
containers.
d) Trace metal vacutainers are available commercially.
e) For random urine sample and tissue biopsy samples a plain plastic container with no added
preservatives is preferred
f) For 24-hour urine collections, it is important that the urine collections should not be made into
disposable fiber or stainless steel containers, and polyethylene bottles should be used with no
chemical additives. On receipt in the lab sample volume should be noted and aliquots stored at
4-150C before analysis.

• Zn, Mg, and Mn are at much greater concentration in red cells than in
plasma and, therefore separation of plasma or serum from the cells
should be completed within about six hours.
• Blank tubes should always be checked before any collection system is
used.
• If samples are to be referred to a specialist laboratory for analysis it is
useful to include a blank tube together with the specimen(s).

General considerations…….
• Analytical methods:
a) Method must be sensitive, specific, accurate, precise and relatively fast
b) Detection limits are very important considering measurements are in ng/g to
ug/g range and in practice, the concentration of the trace/ultratrace element
must be at least 10 times the detection limit of the method ensuring sufficient
accuracy and precision
c) Methods include spectrophotometry, atomic absorption
spectrophotometry(AAS), inductively coupled plasma optical emission(ICP-
OES), inductively coupled plasma mass spectrometry(ICP-MS)
d) Other less commonly used methods include neutron activation analysis(NAA),
x-ray fluorescence(XRF) and electrochemical methods like anodic stripping
voltammetry(ASV). NAA requires a nuclear irradiation facility and ASV requires
completely mineralized solutions for analysis (time-consuming process)

Quality assurance considerations
• An effective quality assurance scheme for trace or ultratrace element analyses
requires incorporation of the following into each batch of analyses:
a) reagent blanks,
b) replicate analyses to assess precision,
c) calibrators of the trace elements of interest in the expected concentration
range of the specimens analyzed, and
d) a control or reference solution with known or certified concentrations of the
trace elements to be determined to assess accuracy and batch-to-batch
precision. The reference material should be of the same matrix type and
contain approximately the same amounts of analyte as the specimens.
• It is also essential that trace element laboratories participate in external quality
assessment programs

TRACE METALS 2023.pdfjesmiemma89@gmail.com.un

ZINC (Zn)
• Zinc is second to iron as the most abundant trace metal.
• It is found in all tissue types and fluids, as well as being the most abundant
intracellular trace element.
• It is an important metal cofactor, essential for the functioning of over 300
enzymes that are involved in major metabolic pathways. E.g. carbonic
anhydrase, alcohol dehydrogenase, alkaline phosphatase.
• In addition, it participates and regulates nucleic acid and protein synthesis,
and is required for the functioning of at least 3000 transcription factors.
• Zinc performs many roles in the physiology of the human body including
catalytic, regulatory, and structural roles. Since Zn is required for anabolic
processes, zinc deficiency has a significant effect on growth, tissue
integrity, and wound healing

Zn
• In plasma, zinc is bound to albumin and α-2-macroglobulin.
• There is no dedicated zinc store in the body and about 10% of intracellular
zinc in the liver as well as some other tissues is available as a functional
pool that exchanges with the plasma pool, and is important in maintaining
the plasma concentration that undergoes a rapid turnover.
• Homeostasis is maintained through interactions between the SLC30 [SLC is
solute-linked carrier] (ZnT) family of transporters and the SLC30 (Zip) family
of transporters. The former promote zinc efflux from cells, decreasing
intracellular zinc concentrations, while the latter promote zinc influx,
resulting in increased intracellular zinc concentrations.

Zn
• Absorption of dietary zinc occurs in the duodenum and proximal jejunum
and is facilitated by the SLC39A4- (ZIP4) encoded zinc transporter.
• Absorbed Zn is transported to the liver by the portal circulation, where
active incorporation into metalloenzymes and plasma proteins, such as
albumin and alpha-2-macroglobulin, occurs. Blood plasma contains less
than 1% of the total body content of Zn and lies within a narrow
concentration interval
• About 80% of plasma Zn is associated with albumin, and most of the rest is
tightly bound to alpha-2-macroglobulin. The Zn on albumin is in
equilibrium with plasma amino acids (mostly histidine and cysteine), and
this small (,1%) ultrafilterable fraction may be important in cellular uptake
mechanisms

Zn
• The total adult body content of Zn is about 2 to 2.5 g, and the metal is
present in the cells of all metabolically active tissues and organs.
• About 55% of the total is found in muscle, and approximately 30% in bone.
• The prostate, semen, and the retina have particularly high local
concentrations of Zn.
• Almost all Zn in RBCs is present in the form of carbonic anhydrase, so that
RBC Zn concentration is about 10 times higher than in plasma.
Haemolysates normally have about
• Fecal excretion includes both unabsorbed dietary Zn and Zn resecreted into
the gut. The total amount normally equals the total dietary intake and is on
the order of 10 to 15 mg/day in healthy populations. In contrast, urine
output of Zn is normally only about 0.5 mg/day (7.6 mmol/day), but this
can increase markedly during catabolic illness

Zn
• Absorption can be inhibited by iron as well as by fiber and phytate, which
explains why the bioavailable fraction is lower in vegetarian diets that are rich in
phytate. Therefore vegetarians need 50% more zinc than nonvegetarians.
• In the United States, Daily Reference Intake for zinc is 11 mg/day for men and
8 mg/day for women.
• Increased amounts are required during pregnancy and lactation.
• Citric acid can enhance zinc absorption.
• Homeostasis is maintained by intestinal absorption, gastrointestinal (GI)
excretion, urine excretion, and cellular retention. If dietary zinc is low or there is
poor absorption, excretion is diminished and circulating insulin-like growth factor-
I (IGF-I) is diminished, with resultant reduction in growth. If zinc is present in
excess, chelation by metallothionein occurs. In general, zinc excess has minimal
medical consequences while deficiency is quite detrimental.

Zn deficiency
• Can result from inadequate intake, poor absorption, increased loss, or increased
demand. The presenting symptoms of zinc deficiency are most obvious in the
• Integumentary (severe acral and perioral dermatitis, alopecia, abnormal nails,
nonhealing ulcers, delayed wound healing),
• Gastrointestinal (diarrhoea) ,
• Immune (recurrent infections) and
• Skeletal systems (poor growth) but can also involve
• Central nervous system (CNS; impaired cognitive function, altered sense of smell
and taste, depression),
• The visual system (nightblindness, photophobia, blepharitis, conjunctivitis)
• Reproductive system (decreased testosterone levels and fertility) and
• Foetal growth and development, if deficiency is present during pregnancy.

Zn deficiency in children
• It is known that the Zn in human breast milk is effciently absorbed
because of the presence of factors such as picolinate and citrate.
However, the total quantity of Zn in breast milk is related to maternal
nutritional status, and a physiologic decline in the Zn content of
“mature milk” is noted after about 6 months’ lactation

Zn deficiency(in children)
• Zinc deficiency may also occur as a result of inborn errors involving decreased function of zinc
transporters.
• Acrodermatitis enteropathica results from dysfunction of ZIP4, the major zinc transporter in the
intestine and insufficient zinc uptake since, there are no real zinc stores in the body.
• Infants are usually asymptomatic at birth but develop symptoms after breast feeding is
discontinued. ZIP4 is encoded by the SLC39A gene and forms a channel on enterocyte apical
cell membranes.
• The initial symptom is usually a severe erythematous rash, which is often most prominent in the
diaper area. The rash is complicated by intermittent diarrhoea and failure to thrive, if the
condition goes unrecognized.
• Infection can be a severe and life-threatening consequence of prolonged zinc deficiency.
• Zinc deficiency in breast-fed babies can also present with a similar picture. This is commonly seen
in premature infants as the premature gut has reduced zinc uptake capacity and the infant has
increased zinc needs.
• In other cases, this results from reduced zinc levels in breast milk secondary to heterozygous
variants in the SLC30A2 gene.

Zn excess
• Generally considered to be a benign condition and elevated plasma zinc levels
with genetic inheritance have been described.
• The first, autosomal dominant hyperzincaemia without symptoms has been
described in one family and appears to be benign.
• It is hypothesized that this condition is the result of increased binding of zinc to
an altered albumin molecule.
• The second, hyperzincaemia with hypercalprotectinaemia results in extremely
high plasma zinc levels but relative zinc deficiency with concomitant persistently
elevated C-reactive protein, anaemia, arthritis, hepatosplenomegaly,
and recurrent infections. The pathophysiology of this disorder is thought to be
related to very high concentrations of calprotectin, the major zinc binding
protein of phagocytic cells, and that these high levels lead to overly robust and
harmful inflammation along with decreased bioavailability of zinc. No gene has
been yet been identified as causative.

Laboratory assessment
• Plasma zinc determination, though insensitive to dietary zinc intake and
subject to a variety of influences, it remains the most widely used
laboratory test to confirm severe deficiency.
• It is also used to monitor adequacy of zinc provision, especially if
interpreted together with changes in serum albumin and the APR. No
laboratory procedures are established for clearly identifying populations
with marginal zinc depletion.
• The clinical and biochemical responses to zinc supplementation are
therefore used to postulate a marginally zinc-depleted state.
• Plasma Zn levels exhibit both circadian and postprandial fluctuations.
Decreased after food and higher in the morning than evening.

Lab assessment
• Plasma samples are preferred to serum for zinc analysis because of possible zinc
contamination from erythrocytes, platelets, and leukocytes during clotting and
centrifugation(5-15% higher)
• Plasma zinc concentrations are most commonly measured by FAAS though
spectrophotometric methods are available. Care has to be taken in controlling
preanalytical factors that will lower plasma zinc independently of dietary intake.
These include collection of sample in relation to (1) meals, (2) time of day, and (3)
use of steroid-based medications, such as the contraceptive pill. Any cause of
hypoalbuminemia will also lower plasma zinc. Therefore it is good to measure it
together with plasma albumin and plasma CRP or any other APR marker
• Reference interval for clinical guidance 80-120ug/dl (12-18umol/l)
• Fasting morning <70ug/dl (0.7umol/l) on more than one occasion requires further
investigation. If < 50ug/dl (5umol/l) it suggests deficiency
• Urine Zn excretion is in the range 0.2-1.3mg/24h (3-21umol/24h)

Lab assessment
• Blood cell Zinc: It has been suggested that the zinc content of white cells
and platelets better reflects tissue zinc. The zinc content of neutrophils,
lymphocytes, and platelets has been shown to decline more rapidly than
plasma zinc in experimental studies of zinc depletion in humans. However,
the relatively large volume of blood required and problems with
contamination make application to patients in the hospital or to population
surveys difficult.
• Zinc in hair: Low hair zinc has been associated with poor growth in
children. However, variables such as hair growth rate and external
contamination from hair dyes and cosmetics has caused inconsistent
results
• Zinc-dependent enzymes: Despite the large number of zinc
metalloenzymes that have been identified, no single enzyme assay has yet
found acceptance as an indicator of zinc status

Lab assessment
• Metallothionein: Its determination in red cell and MT MRNA in
circulating monocytes is considered of probable value because it falls
in Zn deficiency. No clinical use yet though as it has not been
confirmed by a large-scale investigation of depleted population.
• Urine Zinc: There is a slight fall ln the urinary excretion of zinc during
dietary deficiency. Difficulties of sample contamination during
collection make this of limited practical value. However, increased
urinary loss in the severely injured catabolic patient is important.
Urine output increases with amino acid infusion during total
parenteral nutrition(TPN)

Treatment of Zn deficiency
• For infants with zinc deficiency associated with breastfeeding, dietary
supplementation will result in rapid improvement and the condition
resolves when breast feeding is discontinued or the diet is advanced.

COPPER (Cu)
• It an essential metal required for growth and development.
• It is associated with a number of metalloproteins including cytochrome oxidase,
superoxide dismutase (SOD), tyrosinase, dopamine hydroxylase and lysyl oxidase and is
required by all tissues for proper cellular metabolic function.
• Control of its levels is critical to avoid deficiency or excess, both of which are detrimental.
• Copper can adopt both an oxidized (Cu2+) and reduced (Cu+) configuration and hence, is a
catalytic cofactor in oxidation–reduction (redox) reactions and is vital for proper
functioning of enzymatic pathways involved in energy production and antioxidant
activity.
• The copper content of food is variable and is affected by applications to crops of copper-
containing fertilizers and fungicidal sprays and the use of copper-containing cooking
vessels. The metal is most plentiful in organ meats, such as liver and kidney, with
relatively high amounts also being found in shellfish, nuts, whole grain cereals, bran and
cocoa-containing products

Cu
• Copper absorption occurs mainly at the small intestine. The extent of small intestinal
copper absorption varies with dietary copper content and is around 50% at low copper
intakes (<1mg Cu per day) but only 20% at higher intakes (>5mg Cu per day).
• Gastric uptake has been shown to occur to a lesser extent as well as inhalation and skin
absorption
• Absorption is reduced by other dietary components, such as Zn (via metallothionein),
Mo, and Fe, and increased by amino acids and by dietary sodium
• Absorbed copper is transported to the liver in portal blood bound to albumin, where it is
incorporated by the hepatocytes into cuproenzymes and other proteins and then
exported in peripheral blood mainly as caeruloplasmin to tissue and organs.
• Although two thirds of the 80 - 100 mg total body copper content is located in the
skeleton and muscle, the liver is the key organ in copper homeostasis.
• Caeruloplasmin is a positive APR and increases during infection and after tissue injury. A
smaller amount of copper in plasma (<10%) is bound to albumin.

Cu
• Caeruloplasmin is also increased in pregnancy and during the use of
oral contraceptives leading to a rise in serum copper concentration
• A significant amount of copper is excreted via bile into faeces daily
and patients with cholestatic jaundice or other liver dysfunction are
at an increased risk for copper accumulation due to failure of
excretion.
• Cu losses in urine and sweat account for <3% of dietary intake

Functions of Cu
• Copper is a catalytic component of numerous enzymes and is also a structural
component of other important proteins in humans, animals, plants, and microorganisms.
• Energy Production: Cytochrome c oxidase is a multisubunit complex containing copper
and iron. Located on the external face of mitochondrial membranes, the enzyme
catalyzes a four-electron reduction of molecular oxygen, which is necessary for ATP
production.
• Connective Tissue Formation: Protein-lysine 6-oxidase (lysyl oxidase) is a cuproenzyme
that is essential for stabilization of extracellular matrixes, specifically the enzymatic
crosslinking of collagen and elastin. The enzyme is highly associated with connective
tissue and located in the aorta, dermal connective tissue, fibroblasts, and cytoskeleton of
many other cells.
• lron Metabolism: Copper-containing enzymes-namely ferroxidase I (caeruloplasmin),
ferroxidase II, and hephaestin in the enterocyte oxidize ferrous iron to ferric iron. This
allows incorporation of Fe3+ into transferrin and eventually into hemoglobin

Functions of Cu
• Central Nervous System: Dopamine monooxygenase (DMO) is an enzyme that requires
copper as a cofactor and uses ascorbate as an electron donor. This enzyme catalyzes the
conversion of dopamine to norepinephrine, the important neurotransmitter. Monoamine
oxidase is a copper-containing enzyme that catalyzes the degradation of serotonin in the
brain.
• Melanin Synthesis: Tyrosinase is a copper-containing enzyme that is present in
melanocytes and catalyzes the synthesis of melanin.
• Antioxidant Functions: Both intracellular and extracellular SODs are copper- and zinc-
containing enzymes, able to convert superoxide radicals to hydrogen peroxide, which is
subsequently removed. Ceruloplasmin also binds copper ions and thus prevents
oxidative damage from free copper ions, which generate hydroxyl radicals.
• Regulation of Gene Expression and lntracellular Copper Handling: Metallothionein
synthesis is controlled by copper-responsive transcription factors, and this protein is
important in regulating the intracellular distribution of copper. Additional specialized
proteins act as "copper chaperones" to deliver copper to intracellular sites and prevent
oxidative damage by free copper ions

Functions of Cu
• lnborn Errors of Copper Metabolism: Menkes syndrome is caused by a defective
gene that regulates the metabolism of copper in the body. Wilson disease is
inherited as an autosomal recessive trait having a defect in the metabolism of
copper, with accumulation of copper in the (1) liver, (2) brain, (3) kidney, (4)
cornea, and (5) other tissue. Copper-transporting P-type ATPases, known as
ATP7A and ATP7B, are essential factors in maintaining copper balance. Impaired
intestinal transport of copper caused by a mutation in the ATP7A gene leads to
the severe copper deficiency disease seen in Menkes syndrome. A defect in the
ATP7B gene affects both incorporation of copper into caeruloplasmin and copper
excretion via bile, and is the basis of Wilson disease
• Angiogenesis: Cu plays an important role in promoting angiogenesis and this
knowledge has triggered new nutritional models of cancer therapeutic
intervention
• Requirements and reference nutrient intake: The recommended dietary intake for
adults is 0.9 mg/day. The tolerable upper limit is 10 mg/day.

Extra information
The majority of dietary copper absorption takes place in the duodenum via the high affinity transporter, hCTR1 (human
copper transport protein 1), in the basolateral membrane of the intestinal epithelial cell. Transport is regulated by the intracellular C-
terminal domains of the homotrimer while actual transport is mediated by the extracellular and transmembrane domains. It is
postulated that CTR1 is needed for the release of dietary copper from subapical vesicles for delivery to copper chaperone proteins,
mitochondrial cytochrome C oxidase, and Cu-ATPases. Reductases in the apical membrane are necessary for the reduction of Cu2+ to
Cu+, the ion that is recognized by the transporter. Divalent metal transporter I and endocytic/pinocytic processes are postulated to be
involved in the transport across the apical membrane. A low-affinity transporter, CTR2, has also been identified, the exact function of
which, has not been elucidated but likely facilitates movement of copper across the apical (luminal) membrane. Cu-ATPase ATP7A is
then responsible for the transport of copper into intracellular vesicles and secretion of copper across the basolateral membrane into
the bloodstream. Copper is then transported in the blood bound to albumin, caeruloplasmin, transcuprein, and low molecular weight
copper–histidine complexes for delivery to tissues. It is readily taken up by hepatocytes via hCTR1. After uptake, it is stored via
chelation by metallothionein, is bound to chaperone proteins for intracellular trafficking to copper-requiring enzymes or is bound to
reduce glutathione. ATP7B is necessary for the efflux of excess copper, particularly in the liver. It is also postulated to result in copper
sequestration and storage in other tissues, such as in the kidney and intestine. Both ATP7A and ATP7B are localized perinuclearly and
thought to be targeted to the trans-Golgi network under basal conditions. They both receive copper, which binds to an N-
terminal metal binding site, from a chaperone protein, Atox1. Precise control of copper homeostasis is necessary and disruption
thereof results in disease. Two pools of circulating copper exist. The first pool is copper that is tightly bound to ceruloplasmin, which
includes about 85–90% of circulating copper and is thus not freely available to cells and tissues. The remaining 10–15% of circulating
copper is less tightly bound to albumin and other small molecules in the blood and is more freely available to cells.

Cu deficiency
• Both children and adults can develop symptomatic copper deficiency.
• It can affect the haematologic, immunologic, neurologic, skeletal, and vascular
systems. Furthermore, copper deficiency has been shown to result in
impaired myocardial contractility, cardiac conduction defects, and
neurobehavioral symptoms.
• Premature infants are the most susceptible since copper stores in the liver are
laid down in the third trimester of pregnancy.
• In adults, deficiency is usually found following intestinal resection, bypass surgery
or inappropriate oral zinc supplementation (Zn induction of metallothionein in
the intestinal mucosa which sequesters dietary Cu and blocks its absorption.
• Deficiency usually presents as refractory anaemia or leucopoenia. Neurological
consequences such as spasticity or neuropathy are later complications

Cu deficiency
• Menkes syndrome is a very rare but fatal condition that presents in
infants as growth failure and mental retardation, with lesions of the
major blood vessels and bone disease. A characteristic sign is ‘steely
hair’ (pilo torti). It is an inborn error and the mutation is X-linked and
typically occurs in male infants at 2 to 3 months. Low concentrations
of copper in plasma, liver and brain occur because of impaired
intestinal copper absorption. Local first-line tests would provide
findings of low plasma copper and caeruloplasmin along with
demonstration of pili torti by microscopic examination of the hair

Cu excess/toxicity
• Copper toxicity is uncommon and is most usually due to administration of
copper sulphate solutions. Oral copper sulphate may lead to gastric
perforation. Serum copper concentrations may be greatly elevated.
Treatment is by chelation with penicillamine
• Wilsons disease is a rare inborn error of copper metabolism (autosomal
recessive)
• Wilsons disease is caused by a mutation in the gene ATP7B that codes for a
cation transporting enzyme involved in copper transport. This leads to
(a)impaired biliary excretion and thus deposition of copper in the liver and
(b) deficiency in caeruloplasmin resulting in low copper concentrations
• Presenting symptoms include nonspecific liver disease, neurologic
symptoms, psychiatric illness, hemolytic anemia, skeletal anomalies, and
renal Fanconi syndrome.

Wilsons disease
• All adolescents or young adults with otherwise unexplained neurological or hepatic disease
should be investigated for Wilson’s disease.
• Symptoms are a result of copper deposition in liver, brain and kidney. Copper deposits in the eye
can sometimes be seen as a brown pigment around the iris (the Kayser– Fleischer ring).
• Wilson’s disease is caused by a mutation in the gene ATP7B that codes for a cation transporting
enzyme involved in copper transport.
• Urinary free copper excretion is high and total serum concentrations low
• Confirmation is by measurement of copper in a liver biopsy, which is usually greater than 250
µg/g dry weight in patients with the disease.
• A non invasive 65Cu-oral uptake test is a reliable test for the diagnosis of Wilson’s disease and
available in some specialist laboratories.
• Treatment is by administration of a chelating agent, penicillamine, to promote urinary copper
excretion. Patients are maintained on oral penicillamine for life. Liver transplantation may also be
considered, particularly in young patients with severe disease.

Lab assessment of Cu status
• Plasma copper
• Plasma caeruloplasmin
• Because about 90% of plasma copper is bound to caeruloplasmin, factors that increase
the hepatic synthesis of caeruloplasmin, such as an APR or the oral contraceptive pill will
increase plasma copper independently of dietary copper intake
• In premature infants with liver immaturity and low caeruloplasmin synthesis, plasma
copper values below 30 uglL (<5 umol Cu/L) suggest the necessity for increased copper
input.
• The ratio of immunologically to enzymatically measured caeruloplasmin may be a useful
index of marginal copper depletion. Apocaeruloplasmin increases in blood serum during
copper depletion and this will contribute to the total caeruloplasmin assay, but the
enzymatic activity decreases even in marginal copper depletion
• Clinically, Cu status should be investigated initially by measurement of serum Cu and
assessment of the APR and should be interpreted in light of clinical and drug information

Reference intervals
• For adults, plasma copper is usually in the interval of 70 to 140 ug/dl
(10 to 22 umol/L).
• Values in women of childbearing age and especially in pregnancy are
higher.
• Urine copper output is normally less than 60 ug/24 hr (<1.0 umol/24
h) and values above 200 ug/24h (3 umol/L) are found in Wilson
disease
• A Cu concentration in a liver biopsy sample >250ug Cu/g dry weight
(normally 8 to 40ug/g dry weight) is indicative of Wilsons disease in
the absence of other causes of cholestatic disease

Selenium
• Selenium is required as a prosthetic group for several enzymes, including
glutathione peroxidase which acts an antioxidant together with the tocopherols
(vitamin E)
• Antioxidant system protects membranes and other vulnerable structures from
oxidative attack by free radicals
• The most important biologically active compounds contain selenocysteine where
selenium is substituted for sulphur in cysteine
• Selenocysteine is now considered the 21st amino acid. It is incorporated into
proteins by the specific codon UGA, previously thought to be solely a stop codon
• Selenium enters the food chain mainly as selenomethionine from plants (not
synthesized by animals or humans) that take the element up from the soil but do
not appear to use it. Wheat and other cereal products are a good source of
selenium

• Ingested selenium compounds include selenite, selenite, selenocysteine
and selenomethionine
• These compounds are metabolized largely through selenide and then
converted to selenophosphate which is an important precursor in the
synthesis of selenocysteine
• Intestinal absorption of various dietary sources of selenium is efficient but
not regulated
• About 50% to 60% of the total plasma selenium is present as the protein
selenoprotein P. Approximately 30% of plasma selenium is present as
glutathione peroxidase (GSHPx-3) and the remainder is incorporated into
albumin as selenomethionine.
• Urinary output of selenium is the major route of excretion and reflects
recent dietary intake

Functions of Se
• Thirty or more biologically active selenocysteine-containing proteins are now identified. Some of
the most important ones are listed below
• Glutathione peroxidase: use the reducing power of glutathione to remove an oxygen atom from
hydrogen peroxide and lipid hydroperoxide
• Iodothyronine deiodinase: type I (liver kidney and muscle >90%), II and III (both in pituitary, brain
and brown adipose tissue) isoforms of this enzyme are responsible for conversion of precursor
hormone T4 to the active hormone T3
• Thioredoxin reductases: 3 isoforms catalyse the NADPH- dependent reduction of thioredoxin and
are important in maintaining the intracellular redox state
• Selenophosphate synthethase: is required for the intracellular synthesis of selenoproteins via a
mondelenium phosphate intermediate
• Selenoprotein P: Major selenium containing protein in blood plasma. It may be a transport
protein for the element and it has an antioxidant function
• Selenoprotein W: Found in skeletal muscle that is reduced in concentration in white muscle
disease in animals

Selenium deficiency
• There are important selenium-dependent diseases in farm animals, such as white muscle disease
in sheep and cattle, and myopathy of cardiac and skeletal muscle in lambs and calves
• In humans a range of deficiency states have been identified
• Severe deficiency: symptomatic selenium deficiency has been well characterized in Keshan
disease and nutritional depletion in hospital patients.
• Keshan disease: Conclusive evidence for a role for selenium in human nutrition came with
publication of the results of large-scale trials in China that showed the protective effect of
selenium supplementation on children and young adults suffering from an endemic
cardiomyopathy. This was observed in areas of the country (Keshan region) with low soil selenium
concentrations.
• Nutritional depletion in hospital patients: Inadequate selenium provision in specialized diets used
to treat inborn errors and during long-term parenteral nutrition has led to cases of deficiency.
Symptoms of severe deficiency include muscle weakness. Cases involving cardiomyopathy, which
is usually fatal and resembles Keshan disease, and macrocytosis and pseudoalbinism in children
have been described.

Marginal deficiencies
• Are involved in thyroid function, immune function, reproductive disorders,
mood inflammatory conditions, cardiovascular disease, viral virulence and
cancer chemoprevention
• Thyroid function: Selenium and other trace elements are necessary for
normal thyroid function since the important deiodinase enzymes are
selenoproteins. Endemic thyroid disease inZaire may be related to the
combination of iodine and selenium depletion. Care must be taken because
the stimulation of thyroid hormone metabolism may induce
hypothyroidism
• Immune function: Deficiency of selenium is accompanied by loss of
immunocompetence and this is related to the reduction of selenoproteins
in the liver, spleen, and lymph nodes. Both cell-mediated immunity and B-
cell function are impaired.

• Reproductive disorders: Adequate selenium supply is necessary for
successful reproduction in a variety of farm animals. Male fertility could be
affected by selenium depletion in so far as it is necessary for testosterone
synthesis and maintenance of sperm viability
• Mood: Marginal selenium depletion has been associated with anxiety,
confusion, and hostility, and improvements have been claimed following
supplementation
• Inflammatory: Many conditions associated with inflammation and
increased oxidative stress could be influenced by selenium status. Positive
effects from supplementation studies in arthritis, in pancreatitis, and in
intensive care have been reported.
• Cardiovascular disease: not conclusive

• Viral virulence: An unusually virulent strain of the Coxsackie virus is probably part of the cause of
cardiomyopathy in selenium-depleted regions of China. This is consistent with the seasonal
variations in the incidence of the disease. In laboratory studies, a nonlethal form of Coxsackie B
(CVB 3/O) mutated to a virulent strain when inoculated into selenium-deficient mice, probably as
a result of oxidative stress. Further animal studies have demonstrated that a mild strain of
influenza virus exhibits increased virulence when given to selenium-deficient mice. The relevance
of these studies to humans needs to be established.
• Cancer chemoprevention: Epidemiological surveys have found a link between cancer incidence
and soil selenium content, suggesting a higher incidence of certain cancers in individuals with a
low selenium intake. Large-scale trials in China on people having a high risk for viral hepatitis B
and liver cancer demonstrated that selenium-enriched table salt led to a reduction of liver cancer
incidence of 35%. It now seems likely that selenium supplementation above the minimum dietary
requirement has a role in cancer prevention, particularly in relation to prostatic cancer. However
the large Selenium and Vitamin E cancer prevention trial (SELECT) has concluded the selenium or
vitamin E alone or in combination at the doses and formulations used did not prevent prostate
cancer in the population of relatively healthy men.

Selenium toxicity
• Areas of China and the United States have high amounts of selenium
in soil, and locally produced food contains excess selenium Clinical
signs of selenosis are garlic odor in the breath, hair loss, and nail
damage. The tolerable upper limit has been set at 400 ug/day for
adults and less for children
• Cases of toxicity from self-administered dosages have been reported.
• No antidote yet known.

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