dIN.
CHEM. 2 1/4. 501-520(1975)
Newer Aspects of the Roles of Zinc, Manganese,
and Copper in Human Nutrition
Robert E. Burch, Henry K. J. Hahn, and James F. Sullivan
Advances
in knowledge
of the trace elements zinc,
manganese, and copper are reviewed (151 references),
particularly as related to human metabolism and disorders. The literature reviewed, with few exceptions,
is
that published by December 1973.
Additional
Keyphrases:
absorption,
distribution in the body,
excretion, function, pathological manifestations
of deficiency,
and toxicology of trace elements
#{149}
normal values #{149}
metalloenzymes
#{149}
hormone activity
The primary
purpose
of this review
is to discuss
what is now known about three of the most-studied
essential
trace elements-zinc,
manganese,
and copper-as
these elements
relate to humans.
Early workers used the adjective
“trace”
for those
elements
present
in such small amounts
in living tissues that
they could not be measured
with the methods
available. Thus, the term “trace
element”
was born. As
methodology
improves,
additional
elements
will be
recognized.
If we are, indeed,
what we eat and
breathe
and drink, then it is not unreasonable
to conclude that intake of almost any element
will result in
deposition of this element
riod of time.
Research
Department
in the body for a finite pe-
Service,
Veterans
Administration
of Medicine,
Creighton
University
Hospital,
School
and the
of Medi-
cine, Omaha, Neb.
Address
reprint
requests
to Dr. Burch at the Veterans
Administration
Hospital,
4101 Woolworth
Ave., Omaha, Neb. 68105.
Received Oct. 18, 1974; accepted
Nov. 8, 1974.
Of the trace elements
have been designated
appearing
“essential”
in the body, 10
trace
elements:
zinc, manganese,
copper, iodine, iron, cobalt, molybdenum, tin, selenium, and chromium.
Iodine is intimately associated with thyroid physiology; cobalt is a
component
of vitamin B12; and iron is both a component of hemoglobin
and closely related
to the cytochrome
system.
Of the seven
remaining
essential
trace elements,
zinc, manganese,
and copper
have
been studied
most extensively.
Because
more infor-
mation is available for these latter three elements, we
have arbitrarily
elected to limit our discussion
to
them. Deficiency of an essential trace element results
in a characteristic
deficiency
syndrome
in a manner
analogous
to a specific vitamin
or hormone
deficiency. The deficiency
syndrome
is associated
with specific structural,
functional,
biochemical,
or physiological abnormalities.
These abnormalities,
in turn, are
prevented
or reversed
after administration
of the deficient
element.
Similarly,
toxicity
may result from
excesses of these essential
trace elements.
Just as in
the case of vitamins
A or D or the various hormones;
it does not follow that if a little is good, then more
should be better.
The field of trace elements
is held in ill repute by
some clinicians because the term “trace elements”
frequently
elicits a picture of the lunatic fringe food
faddists.
Unsubstantiated
and
unscientific
claims
and counterclaims
have contributed
to this reputation. We think this reputation
is ill-deserved because
CLINICAL
CHEMISTRY.
Vol. 21, No. 4, 1975
501
of the numerous
conscientious
scientists working in
the field in the past, and because of the influx of new
scientists who have been attracted
into trace element
research because of its exciting potential for new contributions
to medicine.
Zinc
Zinc is ubiquitous
in plants, microorganisms,
and
animals. In those animals that have been evaluated,
zinc has been shown to be an essential trace element.
In growing animals, deficiency
of zinc results in a
characteristic
picture: loss of appetite,
inability to
gain
weight,
skeletal
abnormalities,
parakeratotic
esophageal and skin lesions, hair abnormalities,
and
inhibition of sexual maturation.
Although few studies
have
little
been
performed
doubt
animals.
on adult
that zinc deficiency
Zinc was shown
animals,
there
can be
can also exist in adult
to be an essential
trace
element
for
the rat in 1934 by Todd et al. (132). In 1940 Keilin
and Mann (68) demonstrated
that carbonic anhydrase (EC 4.2.1.1) is a zinc metalloenzyme.
In 1955
Tucker
and
Salmon
(134)
showed
that
swine
para-
keratosis was related to inadequate
dietary zinc. In
the late 1940’s and in the 1950’s Dr. Bert Vallee’s laboratory was engaged in elucidating
numerous
zinc
metalloenzymes
and myocardial
opinion,
as well as the effect
infarction
his scholarly
of liver
on zinc metabolism.
review
(139)
disease
In our
on the physiology
and biochemistry
of zinc marked the real beginning
of biomedical interest in zinc as it relates to humans.
Numerous
excellent
reviews on various aspects of
zinc have subsequently
appeared
(8, 16, 60, 89, 97,
104, 111, 135). One of the more comprehensive
recent
reviews is that of Mikac-Devi
(87).
Absorption.
An adult 70 kg human body contains
1.4 to 2.3 grams of zinc. The average adult ingests 10
to 15 mg of zinc daily and absorbs about 5 mg, primarily from the small intestine.
The amount of ingested zinc that is available
for absorption
is unknown. Similarly, the mechanism of absorption
is unknown. However, absorption
does not take place by
simple diffusion, but seems to be ordered and regulated. For example, Cotzias et al. (26) have shown in
mice given oral or parenteral
zinc loads that there
was an acceleration
of fecal 65Zn excretion after parenteral
administration
of the isotope. Conversely,
65Zn absorption
decreased
if the body’s load of zinc
was increased.
There are at least two proteins
in intestinal
mucosa
that bind zinc (140), but it is not
clearly
evident
that these proteins
are involved
in
zinc absorption.
Evans et al. (37) showed
that zinc
absorption
is inversely
related
to intestinal
mucosal
zinc content
and they suggest that the latter may be
regulated
by the zinc content
of plasma. The mechanism of this regulation
is unknown.
Erythrocyt.es
contain 75-85% (primarily
in the zinc
metalloenzyme,
carbonic
anhydrase),
plasma
contains 12-22%, and leukocytes
contain
3% of the zinc
in whole blood of humans
(139). The amount
of zinc
502
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
in the
ous investigators.
zinc per deciliter.
Parisi and Vallee
transported
zinc
in serum
blood has been studied by numerNormal serum contains 100 izg of
Recent studies on human blood by
(96) indicate that 30-40% of the
is firmly
bound
to a2-macroglobulin.
The remaining zinc is apparently
loosely bound to albumin. Giroux and Henkin (43) have calculated that
1 ig zinc per deciliter of human serum is complexed
with
basis
the amino acids cysteine
and histidine
on the
of in vitro addition
of these amino acids to albu-
min. Isolation of zinc complexes from human serum
or urine is needed to corroborate
these findings.
Distribution.
Zinc is transferred
from plasma to
cells by an unknown
mechanism.
Cotzias and Papavasiliou (27) suggested
that homeostatic
mechanisms
were involved in the distribution
of 65Zn to the various organs
and in the intracellular
distribution
of
65Zn to the various
organelles
within
a given cell.
When liver cells are fractionated
by centrifugation,
zinc is found in the supernate,
nuclei, microsomes,
and mitochondria,
in that order of decreasing
concentration.
Spencer has summarized
several of her
studies on the distribution
of 65Zn in humans (124).
She showed
that 65Zn could be recovered
from the
plasma
within 15 mm after ingestion of the isotope.
The maximal
concentration
in plasma was usually attained at 4 h. The concentration
of isotope in plasma
was higher than in whole blood at 4 h. The opposite
results were obtained
if 65Zn was given intravenously.
After five days the concentration
of Zn
in whole
blood was 2-5-fold
that in the plasma
after ingestion
of the isotope.
Liver, kidney, spleen, intestinal
mucosa, lung, pancreas, thyroid,
pituitary,
testes,
and adrenals
show
rapid uptake
as well as turnover
of Zn.
The turnover rate of 65Zn is relatively
slow in brain, muscle,
and erythrocytes.
Hair and bone have very slow 65Zn
turnover
rates.
Excretion.
The primary
excretory
pathway
for zinc
is the gastrointestinal
tract. The mechanism
of excretion is unknown.
Small amounts
of zinc may be lost
in the urine (300-700 g/24
h) and as much as 1 mgI
liter may be lost in sweat. The amount
of zinc appearing
in the stool is directly
related
to the amount
of zinc in the diet (26). The contribution
of pancreatic secretions
to the zinc appearing
in the stool is unknown.
However,
Sullivan
et a!. (129) have shown
that the zinc content
of pancreatic
secretions
in normal adults is about 1 ug/ml. If we assume a maximum
pailcreatic
secretory volume of 1500 ml and no zinc
reabsorption,
then
the
maximum
contribution
of
pancreatic-juice
zinc to the zinc appearing
in stool
would be 1.5 mg. Thus, on a 15-mg zinc intake, 10 mg
of zinc will be found in stool and a maximum
of 1.5
mg could come from the zinc in pancreatic
secretions.
Functions
of zinc. One of the most intriguing
and
perplexing
problems
in trace element
metabolism
has
been to demonstrate
the molecular
lesion(s)
involved
in the deficiency
state.
Although
much
is known
about
the cellular
and metabolic
effects
of zinc, the
mechanism(s)
involved in the production
of the syndrome of zinc deficiency in animals is unknown at
this time.
Because zinc is an integral component
of various
metalloenzymes
and, along with other metals, can activate a wide variety of enzymes, it would seem to follow logically that the syndrome of zinc deficiency is
due to a decrease in the activity of essential zinc-containing enzymes. Indeed, this hypothesis
may ultimately prove to be the correct one for explaining deficiency effects.
A zinc metalloenzyme
found in one organism need
not be a zinc-containing
metalloenzyme
in another
species-e.g.,
it does not logically follow from the
identification
of Bacillus
subtilis
neutral protease as
a zinc metalloenzyme
that neutral protease in rat or
pig or man is a zinc metalloenzyme.
A good example
of this point is the work of Scrutton
et al. (116, 117),
1961 when Prasad et al. (106) described
a group of
18- to 20-year-old Iranian males with iron deficiency
anemia, hepatosplenomegaly,
geophagia, hypogonadism, and dwarfism. These authors suggested zinc deficiency as the cause of this syndrome.
They theorized that the ingestion of clay resulted in chelation
of both iron and zinc. Although not conclusive, it is of
interest that Smith and Halsted (123) have shown recently in rats that treated Iranian clay was protective
for zinc-deficient
animals. Unfortunately,
untreated
clay could not be used in this study because it caused
urolithiasis.
Subsequently,
Prasad et al. (107) described a group of 16- to 19-year-old Egyptian males
with iron deficiency
anemia,
hepatosplenomegaly,
dwarfism,
and hypogonadism.
These patients
had
who showed
appeared from the blood at a faster rate and into a
smaller pool in the dwarfs as compared to controls,
and that it was also excreted more slowly by the
dwarfs than by controls. Additionally,
these dwarfs
had decreased concentrations
of zinc in plasma, hair,
and sweat. These studies seem to indicate that these
dwarfs were zinc deficient.
In fact, the conclusion
that they were zinc deficient would be indisputable
if
additional
information
about the age, size, and clinical status of the control or normal individuals
had
been provided. The data for these, as well as for numerous other papers from this group, have been summarized by Prasad (105).
Finally, Sandstead
et al. (112) have studied 11 of
the hypogonadal
dwarfs described
by Prasad who
were treated with various dietary regimens. A nutritous, high anithal-protein
diet resulted in an average
growth rate of 1.8 inches per year when the diet was
supplemented
with iron in four of these individuals.
Nine patients receiving zinc supplementation
of the
diet grew at a rate of 5.0 inches per year. Two additional patients left the study early and returned
to
their village, and did not grow during the subsequent
395 and 300 days, respectively.
In addition to growing, patients
receiving zinc developed
sexually as
manifested
by growth of genitalia and development
of secondary sex characteristics.
Quite logically, Sandstead
et al. (112) have suggested that the endocrine abnormalities
in these individuals are those of hypopituitarism
on the basis of
poor growth, hypogonadism,
decreased pituitary corticotropin reserve, abnormal glucose-tolerance
curves
(flattening
and delayed absorption
were common),
and increased sensitivity to intravenous
insulin. Recently Coble et al. (17) studied the endocrinological
status of 18 rural Egyptian boys who were prepubertal, ages 15-20 years, and who were below the third
percentile in height and weight for U. S. children and
were short compared
to other rural Egyptian
boys.
Additionally,
they studied eight pubertal boys, ages
14-18 years, whose mean height and weight were
comparable
to, or greater than that for rural Egyp-
that
pyruvate
carboxylase
purified
from
chicken liver was a manganese metalloenzyme
whereas pyruvate carboxylase
purified from baker’s yeast
was a zinc metalloenzyme.
In no way are we deprecating the numerous studies of enzyme activity in relation to zinc deficiency;
however, we are suggesting
that these results be viewed cautiously
because extrapolations
across species lines cannot be readily
made.
A review of a number of investigations
indicates
that zinc may be intimately
involved in protein,
RNA, and DNA synthesis (4). More recent studies by
Slater et al. (122) showed that zinc is tightly bound
to DNA polymerase
(EC 2.7.7.7) from E. coli and
from nuclei of the sea urchin. Scrutton
et al. (118)
demonstrated
that DNA-dependent
RNA polymerase (EC 2.7.7.6) from E. coli is a zinc metalloenzyme.
The general applicability
of these observations
must
await purification
of these enzymes and zinc analysis
in mammalian
systems.
Other areas where zinc might function
have received little attention.
A very perplexing problem in
our minds is the rapidity of onset of symptoms after
an experimental
animal is put on a zinc-deficient
dietary regimen (89). Just as perplexing
is the disappearance
of symptoms
after zinc supplementation.
The rapidity of the process could be explained by diminished activity of a key enzyme. However, it seems
just
as likely
that
other
processes
may
be involved.
Such a process might be control of the transport
of
metabolites
across membranes.
Studies of this sort
have not been done, to our knowledge. Another area
that has been ignored is the effect of zinc deficiency
on other trace elements. Burch et al. (in press) have
shown that zinc deficiency is associated with changes
in tissue copper, magnesium,
manganese,
and selenium in pigs. Both of these possibilities
require further
comprehensive
studies.
Zinc Deficiency
Zinc
zinc that
in man. The seed of interest
in
Vallee sowed in 1959 bore its first fruit in
deficiency
parasitic
infestation
with hookworm
and schistosomiasis. However,
they were not geophagics.
Intravenous
administration
of 65Zn indicated
that the isotope dis-
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
503
tian males. Themean
tions in prepubertal
boys. Administration
produced
plasma testosterone
concentraboys were less than in pubertal
of human choriogonadotropin
an appropriate
response
(doubling
plasma
of both groups.
the human
zinc.
the
depend
Wound
healing.
healing
process
on adequate,
The
available
beneficial
effect
dietary
of zinc on
in human
surgical
wounds
was
et al. (101, 102). These authors
testosterone
values) in three-fourths
Prepubertal
boys had lower plasma lutropin (luteinizing hormone) concentration
than did pubertal boys.
Hypothalamo-pituitary-adrenocortical
axes
were
normal in both groups, in contrast to the findings of
Sandstead
eta!. (112). Increased sensitivity to insulin
was confirmed
in both groups of boys, who also
showed an impaired somatotropin
(growth hormone)
suggested by Pories
studied the healing rate of marsupialized
pilonidal sinuses in 10 young airmen receiving 150 mg of elemental zinc daily (220 mg of ZnSO4 7H20, USP, three
times daily). Ten airmen undergoing the same procedure without zinc supplementation
served as controls. In both groups the healing rate was identical
for the first 15 days, at which time wounds were 83%
response
healed.
to hypoglycemia.
Ronaghy et al. (108) confirmed
the effect of zinc in
accelerating
the rate of sexual maturation
in sixty 12to 14-year-old Iranian boys who were below the third
percentile
for growth. However, they could not confirm an effect of zinc on growth. Carter et al. (12)
were unable to show any effect of zinc or iron on
growth or sexual maturation
in a double-blind
study
on 279 Arab boys. However, they used two-thirds
the
dose of zinc and one-third
the dose of iron used in the
earlier studies. Additionally,
diets of these boys were
limited in calories, vitamin A, riboflavin, and possibly
in calcium and ascorbic acid.
Although their work seems to have received little
attention,
the studies of Coble et al. (18, 19) have cast
some doubt on Prasad’s original findings. Coble et al.
returned
to the Kharga oasis in outhwest
Egypt
three-and-one-half
years after the original study (described in references 105, 107) had been carried out.
They found that the original subjects had sexually
matured
and had attained
a stature comparable
to
adult Egyptian villagers. However, their plasma zinc
concentrations
were
zinc values
were found
with retarded
sad’s criteria
unchanged.
Further,
in a new group
maturation
(these
of “hypogonadal
low plasma
of subjects
individuals
dwarfs”);
fit Prahowever,
randomly
selected subjects with normal growth and
development
had low plasma zinc concentrations,
comparable
to those for the children with retarded
development.
It seems likely that other limiting nutritional factors were involved in these studies. A recent study (47) seems to lend support to the original
observations
relating zinc deficiency to poor growth
in humans. Hambridge
et al. (47) demonstrated
low
concentrations
of zinc in the hair of children from
middle-
and
children
upper-income
had poor growth
families
in Denver.
These
and some had diminished
taste acuity.
Zinc supplementation
resulted
in improved appetite,
growth, and taste.
Evaluation
of the available
information
leads us to
the conclusion that insufficient
data are available to
form absolutely
valid conclusions.
A carefully controlled study under metabolic ward conditions along
with
data
and
sufficient
information
on
control
sUbjects is clearly needed before final conclusions can
be drawn about zinc deficiency
and hypogonadal
dwarfism.
However, preliminary
evidence indicates
that sexual maturation,
growth, and development
in
504
CLINICAL
CHEMISTRY.
Vol. 21. No.4,
1975
.
Healing
was
complete
in
the
zinc-supple-
mented group by 45.8 days vs. 80.1 days in the control group. Thus, zinc therapy seemed to enhance
wound healing at the stage of epithelialization.
Unfortunately,
serum zinc was not measured
in these
patients.
The above observation
sequently
become
quite
was exciting,
controversial.
but it has subA number
of
studies in humans and animals
support or refute the original observations
on the beneficial effects
of zinc on wound healing. These studies have been reviewed by Chvapil et al. (16). Perhaps the controversy is best exemplified
by the study of Barcia (6), who
repeated the original study carried out by Pories et
al. (101, 102) and could not demonstrate
a beneficial
effect of zinc on healing rate in marsupialized
pilonida! sinuses.
studies
It must
that the status
be concluded
from
the
of zinc supplementation
various
in the
healing process of normal individuals
is not known.
The studies
of Sandstead
and Shepard
(113) and
of Oberleas
et a!. (91) clearly indicate that zinc defi-
ciency in the rat is associated with decreased tensile
strength in surgical wounds. Similarly, several studies by Hsu et al. (57, 58, 125) have shown decreased
protein synthesis and collagen synthesis in skin from
zinc-deficient
animals as well as decreased DNA synthesis in skin of surgically wounded
animals. (Ed.
note: see one such paper
in this issue.)
Thus, although zinc supplementation
to improve wound healing in zinc-repleted
individuals
is of dubious value,
the experimental
evidence supports the value of zinc
supplementation
in zinc deficiency. Improved wound
healing in zinc-deficient
patients after zinc therapy
has been demonstrated
in the case of chronic skin ulcers, burns, and in debilitated
patients. These studies
have been reviewed by Chvapil et al. (16). Again,
carefully
controlled
studies are needed. Most of these
human studies
require
better experimental
protocols
and better measurements.
As frequently
happens in any field, relevant studies may go unnoticed.
Before leaving the subject of
wound healing we would like to call attention to some
very elegant recent studies and theories of Chvapil,
which
he has succinctly
summarized
(16).
He
suggests that zinc functions to stabilize membranes,
perhaps
structures.
by decreasing
He further
lysyl oxidase
lipid peroxidation
of these
suggests
that
zinc inhibits
in vitro. This copper-dependent
enzyme
is involved in the formation of aldehyde groups that
are necessary for the covalent cross-links in collagen
polypeptides.
Such studies seem to offer important
new insights for future trace-metal
studies on wound
healing. They suggest that the salutary effect of zinc
in wound healing may be related to this membranestabilizing
effect. Thus, cell damage would be decreased.
Sensory
abnormalities.
Henkin et al. (48, 53) described a syndrome characterized
by decreased taste
acuity (hypogeusia)
and decreased
olfactory acuity
(hyposmia),
which may also be associated with perverted taste (dysgeusia)
and perverted
smell (dysomia). Of the 35 patients described initially, all had
hypogeusia
but the presence of dysgeusia, hyposmia,
and dysomia was not universal. Because of the above
symptoms,
these patients often developed anorexia,
weight loss, and psychological
difficulties,
which occasionally were severe enough to result in marked depression with suicidal tendencies.
Earlier work done in Henkin’s laboratory had indicated that abnormalities
of taste and smell were associated with hepatitis.
Similar disorders
found in
women during the first trimester
of pregnancy were
related to zinc abnormalities.
Therefore, zinc supple.
mentation of patients with idiopathic hypogeusia was
tried empirically
and was quite successful
in relieving
or markedly
improving
the symptoms.
Henkin
(52) had previously
demonstrated
that
treatment
with copper, zinc, or nickel resulted
in improvement
in patients
with several
taste
defects.
Thus,
the mechanism
of development
of the syndrome
of idiopathic
hypogeusia
is not known,
but
trace elements
seem to be involved.
Of special interest along these lines is the observation
(47)
that
young
children
with poor growth,
low zinc in their
hair, and decreased
taste acuity
responded
by all
three criteria to zinc supplementation.
Also of great interest
is the fact that 51% of the 35
patients
with idiopathic
hypogeusia
had had a recent
respiratory
infection.
Numerous
infectious
processes
are associated
with decreased
serum zinc concentrations (8) via release of leukocyte
endogenous
mediator (vide infra). Infection,
or any other stress, results
in liberation of adrenal steroids, which also seem to
be involved in regulating serum zinc and copper concentrations.
That adrenal steroids are somehow involved in regulation of serum levels of zinc and copper is an inductive conclusion based on several experimental observations.
Some control mechanism
has
to be involved, because concentrations
of zinc and
copper in the sera of fasting persons normally remain
in a relatively narrow range under a wide variety of
dietary
and environmental
conditions.
Recently,
Flynn et al. (41) reported
poor wound healing and
low serum zinc values in 10 patients who underwent
bilateral adrenalectomies
and who were maintained
on 40 to 50 mg of hydrocortisone
daily (about twice
the normal adrenal output per day). Lifschitz and
Henkin
(78) have shown
in carefully
controlled
human studies that both serum zinc and copper undergo circadian variation. There seems to be an inverse relationship
between serum zinc and copper
concentrations
and adrenal steroid production.
Thus,
at times when the adrenals would be producing large
amounts of hormone, the plasma zinc and, copper are
at their lowest values. This relationship
is not absolute, however, because serum zinc and copper reach
their zenith at 10 a.m., when adrenal steroid production is still relatively high. Further,
suppression
of
the adrenal
and of corticotropin
(ACTH)
production
with prednisone
did not diminish circadian variation
in serum copper and zinc. In fact the variation
seemed to be accentuated.
Although
these studies
were brief and involved only small numbers
of patients, they seem to indicate that the circadian variation is accentuated
in the presence of cortisone and is
not due to corticotropin.
Earlier studies by Henkin
(49, 50, 51) indicated
that patients with adrenal insufficiency have increased taste, smell, and auditory
acuity, which reverted to normal with cortisone replacement
therapy.
These findings
are consistent
with the above hypothesis.
Thus, it appears
that
adrenal steroids enhance the homeostatic
control of
serum copper and zinc and are secondarily
involved
in sensory perception.
Obviously much work is needed in this whole area. The entire subject of how trace
elements affect sensory perception
has not been approached experimentally.
Teratogenicity.
Blamberg et al. (10) reported
that
feeding a zinc-deficient
diet to breeding
hens resulted in diminished
hatchability
and gross embryonic anomalies manifested
by impaired skeletal development. Subsequently,
Hurley and Swenerton
(61)
found that extremely zinc-deficient
female rats could
not reproduce. There were marked effects on the estrous cycle and in most instances mating did not take
place. These adult rats apparently
did not show any
other signs of zinc deficiency. Therefore,
female rats
were first maintained
on a marginally
zinc-deficient
diet
and after
mating
were placed
on a severely
zinc-
deficient diet. On day 21 of gestation (22 days is the
normal gestation time in the rat) the pups were delivered by caesarean section and the uterus was examined for resorption of fetuses. Remarkably,
of the 280
implantation
sites in 38 zinc deficient rats, 54% of the
fetuses had been resorbed and 45% of the fetuses had
congenital
anomalies. Thus, 99% of the fetuses had
been affected! Hurley et al. have extended these observations
with numerous
excellent studies, which
have recently been summarized
(61). When the zincdeficient diet was given continuously
from day 0 to
21 of pregnancy, all fetal organ systems were affected
and 90% of fetuses obtained on day 21 had gross congenital anomalies. Even transitory
periods of feeding
the zinc-deficient
diet were teratogenic.
For example,
when the zinc-deficient
diet was given from days 4 to
12, 29% of full-term fetuses had anomalies; and if the
zinc-deficient
diet was given from days 6 to 14, almost half of term fetuses were abnormal.
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
505
The
most
frequent
congenital
malformations
found in fetuses obtained by caesarean section, in decreasing
order of frequency,
were: tail anomalies,
clubbed feet, fused or missing digits (syndactyly),
hy-
particularly
fascinating
because low serum and liver
zinc values as well as hyperzincuria
occur with cirrhosis (139). Some noncirrhotic
alcoholics also have low
drocephalus
revert to normal after abstinence
from alcohol (128)
in contradistinction
to the cirrhotic. Thus, it seems
likely that these alcoholic women, with the added
stress of pregnancy
and poor diet, may have been
zinc deficient.
Unfortunately,
zinc concentrations
were not measured in either the mothers or the children (K. L. Jones, personal communication).
All of
the circumstantial
evidence mentioned
above strongly suggests that zinc deficiency in pregnant humans
may result in congenital anomalies in the children. A
careful study to demonstrate
or to refute such a relationship is sorely needed and long overdue.
Diagnosis
of zinc deficiency.
Up to this point we
have carefully avoided discussing how a diagnosis of
zinc deficiency can be made, because we wanted first
and
hydranencephaly,
urogenital
abnor-
malities,
scoliosis or kyphosis, lung abnormalities,
cleft palate, small or missing eyes, short or missing
mandible, hernias, and heart anomalies (61). Transitory deficiency may result in changes in the proportion of different anomalies seen. For example, feeding the zinc-deficient
diet from days 6 to 14 gives the
following anomalies
in decreasing
order of occurrence: tail anomalies,
lung anomalies,
syndactyly,
urogenital anomalies, and cleft palate.
Although there have been no reports in the literature on human congenital
anomalies
and zinc deficiency in the mother, we believe that several studies
give strong circumstantial
support to such a relationship. Sever and Emanuel (119) have also suggested
that there may be a relationship
between maternal
zinc deficiency and congenital
malformations
of the
central nervous system in man. The prevalence
of
congenital
malformations
of the central
nervous
sys-
tem in a recent World Health Organization
(127) survey of 24 centers in 16 countries showed the highest
rates to be in Belfast, Northern
Ireland; Alexandria,
Egypt; and Bombay, India. The prevalence
of anencephaly in Shiraz, Iran, is also quite high (29). Thus,
two of the four areas in the world with the highest incidence of congenital
anomalies
of the central nervous system are in the countries where Prasad et al.
(105) described
zinc deficiency in humans. This, coupled with the very high incidence of central nervous
system anomalies
found by Hurley in the offspring
delivered
from zinc-deficient
rats, is highly suggestive of a relationship
between zinc deficiency
and
congenital
anomalies
in humans.
Further,
Halsted
(46) recently reported that an individual
was excluded from the original study on zinc deficiency in Iran
(107) because he had had extreme
bony malformations since birth. Additionally,
the report of Hambridge et al. (47) and the review by Sandstead
(110)
clearly indicate that zinc nutriture
may be poorer
and zinc deficiency
more common than previously
believed. Finally, Jones et al. (63) recently reported
on eight children born of chronic alcoholic mothers.
Each child had multiple congenital anomalies.
Mean
birth weight was 2.04 kg (4.5 lbs.) and mean birth
length was 44 cm (17.2 in.). Microcephaly
was present in seven of eight children, and all eight children
were judged to have had prenatal
and postnatal
growth deficiency.
Short palpebral
fissures, present
in all the children, were interpreted
as being secondary to deficient growth of the eyes. Bone and joint
anomalies
were present in five of the children. The
exact status of the mothers is unknown, although two
of the women experienced
delirium tremens during
pregnancy.
Four of the women lost weight during
pregnancy,
two gained weight, and no information
was available for the other two. We find this study
506
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
serum
zinc and
to present
certain
hyperzincuria,
facets
both
of which
and concepts
about
readily
zinc de-
ficiency. How can one make the diagnosis?
It seems
that serum or plasma zinc concentrations
or zinc concentrations
in hair are the only real tools that we
have. Great care must constantly
be exercised in obtaining and processing the sample, to avoid contamination.
Even with these precautions,
difficulties
arise. Hair can readily absorb contaminants
from the
atmosphere
or from hair dyes. How close to the scalp
do you obtain the hair and what length of the hair do
you use? Do the zinc values you obtain represent the
zinc status of the patient here and now or do they
represent
that status weeks ago? Obviously there is
no clear answer to these questions.
Serum zinc concentrations
can be decreased within
a day after a zinc-deficient
diet is administered
to experimental
animals. Thus, analysis of serum appears
to be a far more sensitive tool than hair analysis. But
does a low serum zinc always signify deficiency?
Infections, pregnancy,
surgical procedures,
and stressful situations
such as myocardial infarction or bacterial endotoxin
administration
may also lower serum
zinc. Does this lowering of serum zinc represent deficiency? This question cannot be answered from the
information
available in the literature.
However, we
think
that
an extremely
useful
adjunct
in making
a
diagnosis of zinc deficiency may prove to be the application of Henkin’s (46, 51, 53) tests for taste and
smell when there is any doubt about the significance
of low zinc values.
Serum
zinc values
states.
The literature
in physiologic
and
pathologic
is voluminous
on serum zinc
values in a wide variety of entities and has been reviewed by several authors (8, 16, 87, 105, 139). Patients with cirrhosis frequently
have low serum zinc
and paradoxically
may excrete several milligrams of
zinc in their urine each day. Since these patients have
low hepatic
zinc (139) it seems reasonable
to assume
that they are zinc deficient.
However,
the mechanism
of development
of zinc deficiency
and its role in relation to cirrhosis
are unknown.
It is quite conceivable that zinc deficiency results
in decreased membrane
stability (16), which renders
the liver cells more
susceptible
to injury.
Becking
and
Morrison (7) have recently shown that the metabolism of several drugs is reduced in zinc-deficient
rats
and that there is a concomitant
reduction
of cytochrome P-450 content of hepatic microsomes. All biochemical
lesions
ameliorated
after
zinc repletion.
This observation
could explain why chronic alcoholics with advanced
cirrhosis
are so sensitive
to sedatives and narcotics,
which may readily precipitate
he-
patic coma. Finally, Burch et al. (in press) have
shown in zinc-deficient
pig liver that there is decreased activity of the enzyme, ornithine
transcarbamylase (EC 2.1.3.3), which catalyzes the conversion
of ornithine to citrulline in the urea cycle. Thus, decreased
cirrhotic
activity
of this enzyme
in the
individual
could
contribute
zinc-deficient
to the well-
known phenomenon
of low serum urea nitrogen and
increased blood ammonia. Thus it seems likely that
zinc deficiency
may play
bolic features
of cirrhosis.
Serum
merous
zinc is also diminished
infectious
dotoxins,
a role in some
processes,
after
exposure
of the meta-
surgery,
nu-
to bacterial
en-
or acute
myocardial
infarction.
Kampschmidt et a!. (65, 66) and Pekarek and coworkers
(98,
99, 103) have studied a factor in polymorphonuclear
leukocytes
that is released
in response
to infection,
administration
of bacterial
endotoxin,
or tissue damage. This substance
has been designated
as “leukocyte endogenous
mediator”
or LEM.
Some of the
more recent
studies
in animals
have been reported
have rein man after experimen-
(65, 66, 98, 99, 103) and Beisel and Pekarek
cently
reviewed
the findings
tal infections
in volunteers
(8).
After certain infections,
administration
of bacterial
endotoxin,
or tissue damage,
leukocyte
endogenous
mediator
(LEM) is liberated
from polymorphonuclear leukocytes,
and within an hour there is a net flow
of amino acids to the liver. This is followed shortly by
a decrease
in serum zinc with a concomitant
uptake
of zinc by the liver. Over the next few hours there is
synthesis
of acute phase reactants
by the liver. This
synthetic
process seems to require
insulin and cortisone (62). These acute phase reactants
include fibrinogen, a1-acid
glycoprotein
(orosomucoid),
a2-acute
phase globulins,
and haptoglobin.
Increased
concentrations
of ceruloplasmin
(an a2-acute
phase globulin) result in increased
concentrations
of copper
in
the serum.
LEM also results
in early depression
of
serum iron and seems to be involved
in the production of fever. Determining
whether
LEM and socalled
endogenous
pyrogen
of polymorphonuclear
leukocytes
are the same substance
must await further purification
and characterization
of these products. LEM has been characterized
as a low-molecular-weight
protein
that is heat labile. LEM isolated
from an infected
animal will produce
a response
in a
normal animal.
We suggest that LEM is, in all likelihood;
the fac-
tor responsible
for the heretofore
unexplained
febrile
response,
effects on serum iron, zinc and copper, and
acute phase phenomena
such as increased
erythrocyte sedimentation
rate and increased
haptoglobin
seen in myocardial
infarction,
after surgery and in a
variety of seemingly
unrelated
entities
such as acute
rheumatic
fever or rheumatoid
arthritis.
Thus, one
would anticipate
low serum zinc and iron values and
increased
serum copper values in most situations
associated with inflammation
or cellular damage.
At the present
time we can offer no explanation
for
the low serum zinc values also found in uremia,
leukemia, kwashiorkor,
pernicious
anemia,
or after use
of oral contraceptives.
Zinc Toxicity
Zinc sulfate is a relatively
nontoxic
compound
but,
by no means, can we say that it is innocuous.
To our
knowledge,
long-term
toxicity
studies
are not avail-
able. In contrast, ZnC12 causes tissue necrosis, and inhalation
of zinc oxide fumes results in a chemical
pneumonitis
that may be fatal. Ingestion
of excess
zinc has usually resulted
from storage of food or beverages in galvanized
containers
and results
in fever,
nausea,
vomiting,
and diarrhea.
Long-term
ingestion
of large amounts
of zinc by animals
results
in poor
growth and anemia (8, 87, 135).
Summary
The importance
of zinc in human
nutrition
has
only recently
become apparent.
At the present
time
zinc deficiency
in humans
seems
to be associated
with poor growth and development,
impaired
wound
healing,
and impairment
of sensory perception.
The
association
of congenital
anomalies
with zinc deficiency in the mother
remains
to be evaluated.
However, it appears
likely that this association
will be
demonstrated.
One of the primary
goals of zinc research has to be elucidation
of the mechanisms
in-
volved in development
sociated
of the various
syndromes
as-
with zinc deficiency.
Manganese
The first report
of manganese
deficiency
in man
appeared
in 1972 (31). Until that time it was doubted
that manganese
deficiency
.could occur in humans.
The abnormalities
observed
in that study have made
possible
certain
biochemical
hypotheses
about manganese deficiency
that were previously
inconceivable.
We noted the scarcity of research on the nutritional
role of manganese
in nucleic
acid synthesis,
glucose
utilization
and gluconeogenesis,
intermediary
metabolism, and endrocrine
gland function.
Absorption.
The manganese
concentration
of the
earth’s crust and most plants is relatively
higher than
that of other trace metals,
but the animal
body is
greatly
selective
as to what minerals
it retains,
and
contains
much less manganese
than other elements
that are present
in lesser concentration
in the environment
(88). Therefore,
there must be an efficient
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
507
and specific regulatory
mechanism
for controlling
body manganese concentration.
A 70-kg adult human
body contains
12 to 20 mg of manganese.
It is diffi-
concentrations
cult to assess the average daily manganese
intake of
normal adults
because
it depends
on locality and on
the nature
of the diet. On the basis of average
food
ble changes
in blood manganese
concentrations
associated with specific disorders
have been hindered
by
lack of sophisticated
techniques
for accurate
measurement
of serum
manganese.
Existing
methods
lack simplicity,
sensitivity,
and specificity.
It seems
consumption,
it is estimated
that
human
intake of manganese
mg per day (81).
ranges
the normal
from
adult
0.7 to 22.0
Manganese
content of foods varies greatly. Peterson and Skinner (100) and Schroeder
et al. (115)
found the highest concentrations
in nuts, grains, and
cereals; the lowest in dairy products, meat, poultry,
fish, and seafood. Relatively
high concentrations
of
manganese
were found in soluble
(“instant”)
coffee
and tea and account
for 10% of the total daily intake
(81, 115).
Metabolic
balance studies of manganese
show that
about 20 ig of manganese per day would be retained
under conditions
of minimum
intake. Apparently
this is more than adequate
to prevent a manganese
deficiency
in humans.
Manganese
in natural
food is
in the form of specific complexes and the changes effected
by cooking
animal
purified
are unknown.
experiments
diets
Practically
have been conducted
containing
inorganic
salt
all of the
by use of
mixtures.
This type of diet could alter certain effects on absorption, digestion, transport,
binding to receptors,
and nutritional
value, because the manganese is present as an inorganic
salt and not as an organic
manga-
nese complex.
Human milk is relatively deficient
in manganese
(82, 137). During the first week of life, the infant’s
manganese intake is low (7 gig/day) (114), producing
a negative manganese balance (149). This is followed
by a progressively
increasing
intake of manganese
from infancy to two years of age (82, 114). In spite of
this apparently
diminished
intake during early childhood, a relatively constant concentration
of manganese is maintained
in the liver throughout
life (137,
150). It is unknown
how the infant obtains enough
manganese for its needs and storage sites.
The precise loci and mechanism
of absorption
of
manganese
from the gastrointestinal
tract are unknown. Absorbed
manganese
is transported
as a complex by the globulin,
transmanganin,
which is a spe-
cific manganese-carrying
plasma protein. One manganese atom apparently
binds to more than one globulin molecule (21).
Distribution.
The distribution
and amounts
of
trace elements in human tissues in the United States
and other countries were well documented
by Tipton
et al. (130, 131). Manganese
is widely distributed
in
body tissues and fluids. In the human, the brain, kidney, pancreas,
and liver-in
that descending
order-
show relatively
higher
manganese
concentrations
than do other organs. In animals, the liver, kidney,
and especially the pituitary gland are richer in manganese. Generally,
higher manganese
concentrations
are seen in mitochondria-rich
tissues.
Manganese
508
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
in blood
and serum
vary greatly.
Even
the same investigators
have reported different values
in two separate studies (137). Investigation
of possi-
likely that this obstacle may have been overcome
with the advent of a simplified method for assessing
manganese content of solid tissues and serum by neutron activation analysis (45).
Excretion.
Absorbed
manganese
rapidly appears
in
the bile and is excreted
almost
exclusively
in the
feces, almost none appearing
in the urine. Excretion
also occurs via pancreatic
juice, and reabsorption
into
the lumen of the duodenum,
jejunum,
and ileum. The
excretion
rate through
gastrointestinal
routes
is increased if an animal is given loading doses of manganese or if there is biliary obstruction.
These extra-biliary gastrointestinal
routes are considered
to be auxiliary pathways
for manganese
excretion.
The combination of a principal
excretory
pathway
via bile and
auxiliary
gastrointestinal
excretory
routes
offers an
efficient
homeostatic
regulatory
mechanism
for
maintaining
balanced
manganese
tissue
concentrations (88, 92, 137). Overall homeostatic
regulation of
tissue
manganese
concentrations
is thought
to occur
by selective
excretion
rather
than selective
absorption. In conjunction
with regulatory
excretory
mechanisms, Cotzias
and Greenough
(22) have presented
evidence
that highly
specific
manganese
pathways
exist within the body, but the nature
of these pathways has not been elucidated.
In conclusion,
it is believed
that adequate
intake
plus efficient
homeostatic
control
mechanisms
effectively combine
to prevent
human
manganese
deficiency under
most conditions.
In conditions
where
manganese
requirements
may be increased,
such as
pregnancy,
growth or diabetes,
it has been suggested
(115, 149) that a manganese
deficiency
may develop
in humans.
Function
of manganese.
Even though
manganous
ion is known to be an activator
of many enzymes,
it is
not possible
at present
to correlate
effects on manganese-dependent
enzymes
and the deficiency
state.
However,
a pertinent
investigation
of such a relationship is that of two enzymes
involved
in chondroitin
sulfate
synthesis
(75). The activity
of both of these
enzymes
is decreased
with manganese
deficiency.
Both enzymes
are found in microsomal
fractions
of
epiphyseal
cartilage
homogenate.
These enzymes
are
polysaccharide
polymerase
and galactotransferase,
and they catalyze the following reactions:
(a) polysaccharide
synthesis
from UDP-N- acetyl-galactosamine
and UDP-glucuronic
acid, and (b) incorporation of galactose
from UDP-galactose
into the galactose-galactose-xylose
trisaccharide
that
serves
to
link the mucopolysaccharide
and protein.
Other enzyme activities-bone
phosphatase,
serum
alkaline
phosphatase,
and liver arginase-have
been studied
but have not always correlated
with manganese
deficiency (137).
Many other enzymes
are also known to
be activated by manganese, as recently reviewed (92).
The only now-proven
manganese
metalloenzyme
is
pyruvate carboxylase (EC 6.4.1.1) (116).
Various metal ions are exchanged at the active site
of enzymes, and because manganese may function as
an enzyme activator
it is probable
that it, too, interchanges with other divalent
elements,
and consequently a manganese
deficiency may be associated
with other trace-element
effects.
These
effects
include increased
or decreased
concentrations
of an element or actual exchange
of the element
with manganese in an enzyme.
This possibility
has not been
thoroughly
investigated.
Manganese
seems to be intimately
involved in synthesis of protein,
DNA, and RNA. The DNA-manganese complex was first reported
by Wiberg and Newman (147).
They concluded from its dissociation
constant that manganese
binds to DNA more strongly
than
do other
metals.
Since minute
quantities
of
manganese
were
detected
during
the
isolation
of
RNA and DNA, it was suggested
that manganese
may bear a functional
relationship
to protein synthesis and the transmission
of genetic information.
However, direct evidence
of the in vivo role of manganese
in mammalian
protein biosynthesis
is still limited, although in vitro evidence
indicates
that manganese
is
involved in protein synthesis
(vide in Ira).
Mammalian
cells exhibit
two types of RNA polymerase
activity,
one of which
requires
manganese
(148). Manganese
also affects the DNA polymerase
system.
The RNA-dependent
DNA polymerase
activities
in human
placenta
and rat liver nuclei are
stimulated
predominantly
by manganese.
Manganese
and other cations stabilize
the secondary
structure
of
DNA by their electrostatic
interaction
with the negatively charged
phosphate
group. Extensive
studies relating conformation
and reactivity
to DNA interaction with manganese
ion have recently
been reported
(79).
The results of these investigations
indicate
that
manganese
has an important
role in initiating
protein
synthesis by stimulating
RNA polymerase and DNA
polymerase activities in the mammalian
system.
A study comparing
the effect of manganese
to that
of other ions showed a slight but significant
increase
in protein
biosynthesis,
attributable
to manganese,
in
isolated
rat liver nuclei (146). In kwashiorkor,
there
was a definite
correlation
between
decreased
hepatic
manganese
content
and
decreased
hepatic
protein
content
in the protein-calorie
malnutrition
(72).
Studies
on normal human adults, and some animal
species have shown that relatively
higher concentrations of manganese
are to be found in the adrenals,
pituitary,
and pancreas
than in other tissues.
Manga-
nous ion stimulates
the enzymatic
coupling
of monoiodotyrosine
to form diiodothyronine
in vitro but
the existence
of this coupling
in the intact thyroid
is
questionable.
Rats given manganese
showed a decline
in protein-bound
iodine,
nated
and an increase
thyronines,
reduced
of iodi-
percentage
in iodinated
tyro-
sine (64).
Cortisol
regulates
the manganese-activated
RNA
polymerase
activity
within
mice lymphocyte
nuclei
(69). Corticotropin
and glucocorticoids
cause a decrease
in hepatic
uptake
of manganese
and shift
manganese storage to extra-hepatic
sites (137). Manganese also inhibits
the activation
of adenylate
cyclase (EC 4.6.1.1) by antidiuretic
hormone,
as nanifested by inhibition
of both water flow and sodium
transport.
Current
evidence
suggests
that manganese
and other
system
ions are involved
transmission
it appears
manganese
nervous
control.
Thus
that there is an intimate relation between
and various hormones. The nature of this
relationship
is poorly
sive investigation.
Manganese
in the central
of neurohormone
understood
and requires
inten-
Deficiency
Manganese
deficiency
in humans.
The first recognized case of human
manganese
deficiency
was reported by Doisy (31). While studying
vitamin
K deficiency in a volunteer
under metabolic
ward conditions, it was noted that the patient
had weight loss,
transient
dermatitis,
occasional
nausea and vomiting,
changes in hair and beard color, and slow growth of
hair and beard. Protein
synthesis
seemed to be unaffected. The most striking
finding was hypocholesterolemia.
These findings
were due to the inadvertent
failure
to add manganese
to the purified
diet mix-
ture.
Supporting
evidence
that
this clinical
picture
resulted
from manganese
deficiency
was obtained
by
duplicating
the results
in chicks fed a purified diet
deficient
in both vitamin
K and in manganese.
The
study could not be repeated
in humans
because
sterility seems to be a universal
accompaniment
of manganese deficiency.
However,
this investigation
clearly
demonstrated
that manganese
is an essential
trace element in man.
Experimentally
been produced
induced manganese
in various domestic
deficiency
animals.
has
The
pathogenesis
of symptoms
in this deficiency
is unknown and all attempts
to explain their etiology have
been unsuccessful.
Under conditions
of limited dietary manganese,
homeostatic
mechanisms-especially those controlling excretion-functioned
poorly
and deficiency symptoms appeared.
Teratogenicity.
Studies confirming
manganese
as
an essential nutrient
were reported
simultaneously
by several investigators
(70, 94, 143).
Animals on
manganese-deficient
diets develop numerous
biological and physical
symptoms,
such as decreased
manganese concentrations
in tissues
and milk, suboptimal growth,
decreased
testicular
and ovarian
function, accumulation
of fat, and diabetic-like
glucosetolerance
curves. These findings
vary with the person’s age and the magnitude
and duration
of deficiency. Perhaps
the most remarkable
discovery
was
made by Erway et al. (33), who demonstrated
that a
CLINICAL
CHEMISTRY,
Vol. 21, No.4.
1975
509
manganese
dietary
supplement
fed to pregnant
mutant mice who develop congenital
ataxia would result
in ataxia-free
offspring.
This study demonstrated
for
the first time that supplementation
with an essential
nutrient,
manganese,
can prevent
development
of a
genetically
had
predetermined
not been
phenotype.
abolished,
The mutation
because
offspring
of the ataxia-free mice developed ataxia (vide in Ira).
Ataxia in mice, whether
attributable
to the mutant
gene “pallid”
or to a maternal
dietary manganese
deficiency, is identical;
however,
a high manganese
concentration
(1 mg) in the diet of mutant
mice during
pregnancy
completely
prevented
fect and altered
the mutant
changing the genetic constitution
nese
i
a specific
nutrient
that
the congenital
deexpression
without
(59). Thus, mangaaffects
expression
the mutant gene without altering subsequent
mission of the mutation to future generations
of
transof mice
(32).
Manganese
is necessary
for optimal
growth
in
mice, rats, and other species. Swine, guinea pigs, and
calves do not show impaired
growth with manganese
deficiency
(5). The occurrence
of anorexia
in manganese deficiency
is not well established
(137).
A plausible
biochemical
explanation
for the impaired body growth is not apparent.
Attempts to correlate poor growth with some definitive
chemical
parameter
have contributed
the following
information
(142): (a) Decreased food consumption
is not an adequate criterion of deficiency. (b) Basal metabolic rate
is unchanged.
(c) Gross differences
in nitrogen
absorption
or excretion
were not observed.
(d) Administration
of pituitary
or adrenal
cortical
extracts
caused no weight gain. In a few cases, the injection
of
pituitary
extract
caused
a slight
initial
weight
increase, which leveled off or fell with continued
injections.
(e) Hepatic
arginase activity was decreased,
depending
on the species being investigated.
(I) Impaired growth and faulty bone formation
may be correlated.
Skeletal
abnormalities
have been studied
extensively in manganese
deficiency.
In rats, mice, and
rabbits
the primary
skeletal
effects
are shortening
and bowing of the forelegs.
In rats, these effects are
seen only in the offspring
of manganese-deficient
mothers.
These offspring
exhibited
shortening
of the
radius, ulna, tibia, and fibula from birth to maturity.
In addition,
poor development
of the tibial epiphysis
resulted
in abnormalities
of the knee joint. A high in-
cidence
of ataxia
is also seen in the offspring
manganese-deficient
is irreversible
and is
loss of body-righting
ataxia in manganese
ment of the inner ear.
otoliths
is a result of
polysaccharide
The
skeletal
females.
This congenital
ataxia
characterized
by imbalance
and
reflexes.
The primary
cause of
deficiency
is abnormal
developDefective
morphogenesis
of the
diminished
cartilaginous
muco-
synthesis.
effects
of manganese
deficiency
been studied
extensively
in the chicken.
510
CHEMISTRY,
Vol. 21. No.4,
CLINICAL
of many
1975
have
Chondrodys-
trophy, in humans a congenital abnormality
in development of long bones, has been demonstrated
in
chick embryos
and seems to be identical
to the
human
disorder.
This
chickens
by shortened
wings, globular
contour
dible (“parrot
beak”),
der is experimentally
deficient
diet and may
plementing
the diet
syndrome
is characterized
in
and thickened
legs, short
of the head, shortened
manand high mortality.
The disorinducible
with a manganesebe prevented
simply by supwith manganese.
Perosis,
or
“slipped tendon,” may also occur in manganese-deficient chicks. It is characterized
by enlargement
of the
hocks, short and twisted tibiae, and slipping of the
gastrocnemius
tendon from its condyles.
For further
discussion
of the influence
of manganese on the skeleton,
the reader is referred
to several
reviews (5, 59, 93, 137).
Early
investigators
believed
that impairment
of
the calcification
process
was the primary
factor
in
skeletal
abnormalities,
but subsequent
experiments
demonstrated
that
manganese
deficiency
retarded
endochondral
bone growth
per se, not osteogenesis.
The discovery
that perosis
is related
to changes
in
the mucopolysaccharide
content
of the epiphyseal
cartilage
focused attention
on the chemical
composition of the organic matrix of cartilage
and bone (74).
The galactosamine-containing
polysaccharides
were
drastically
diminished
by manganese
deficiency
in
the chick. Similar
observations
were made in newborn guinea pigs and other species (73, 121, 133). In
addition
ides
to epiphyseal
of guinea
chondroitin
cartilage,
pig rib cartilage
sulfate.
mucopolysacchardecreased,
Hyaluronic
acid
especially
and
heparin
were also decreased
in cartilage
of manganese-deficient animals.
The changes
in polysaccharides
seem
to be specific
for manganese
deficiency.
No other
known nutritional
deficiency
syndrome
resembles
the
pathological
produced by manganese defiin chondroitin
sulfate in manganese deficiency is the result of impaired
mucopolysaccharide
synthesis,
manganese
being a necessary
cofactor for the enzymes involved in chondroitin
sulfate synthesis.
The above studies have demonstrated
that manganese affects the primary sites of chondroitin
sulfate
ciency.
conditions
The decrease
synthesis.
Because
complex
is necessary
nective
tissue,
explanation
these
the
chondroitin
to maintain
findings
for the skeletal
sulfate-protein
the rigidity
provide
of con-
a biochemical
abnormalities
observed
in manganese
deficiency.
We find the association
of manganese
deficiency
in
experimental
animals
and the bony abnormalities
resulting
from the inhibition
of mucopolysaccharide
synthesis
extremely
fascinating
in relation
to the mucopolysaccharidoses
occurring
in humans.
In humans, these diseases
are characterized
by bony abnormalities,
mental retardation,
and accumulation
of
tissue mucopolysaccharides.
Wolff has reported
deform ity and limitation
of motion of the ossicles of the
ear in gargoylism
or an abnormality
(151). Even if manganese
deficiency
of manganese
metabolism
is not
subsequently
shown to be involved in the human mucopolysaccharidoses,
the manganese-deficient
animal
seems to be an ideal model for the elucidation
of the
biosynthetic
pathways
involved in mucopolysaccharide synthesis,
pathways
that are ill-defined
and
poorly understood
at present.
Reproduction.
Manganese has been established
as
an essential
element in reproduction
as well as in
body growth (70, 94, 137, 143). Male rats and rabbits
on manganese-deficient
diets lost their libido and fecundity, and exhibited
testicular
degeneration,
absence of spermatogenesis,
and sterility.
In manganese-deficient
females, there was a delay in the opening of the vaginal orifice. Estrus cycle changes were
diverse. Some cycles were observed to be irregular or
absent, while others were normal. Histological examination
revealed
no ovarian
abnormality.
Similar
studies
in many other species
have shown
impairment of reproduction,
decrease
in litter size, high infant mortality,
and delayed
estrus
and conception
(137). These
investigations
clearly
established
that
manganese
is necessary for normal fertility in the female and that deficiency results in impaired spermatogenesis with subsequent
sterility in the male. Histological
examination
of manganese-deficient
animals revealed atrophy of the testes and an accumulation of degenerating
cells in the epididymis.
The sterility accompanying
manganese
deficiency
has been postulated
to be due to lack of sex hormones
as a result of decreased
cholesterol
synthesis
(31).
Earlier work showed that cholesterol
synthesis was
stimulated
by manganese
(28) and that manganese
is
a required
cofactor
for mevalonate
kinase
(EC
2.7.1.3.6)
(2). For evaluation
of this hypothesis,
synthesis of testicular
cholesterol
and testosterone
needs
to be investigated
in manganese-deficient
animals.
An analogous
irregular estrus
explanation
can be made concerning
cycles and delayed vaginal opening
caused by the lack of female hormones.
Manganese
is
also known to enhance
the binding of estradiol
to nuclear particles;
therefore,
lack of either manganese
or
steroids
may affect the female reproductive
system.
Direct evidence
that lowered
cholesterol
concentrations affect estrogen
synthesis
has yet to be found.
Abnormalities
in glucose utilization.
The hypoglycemic effect seen after administration
of a manganese
salt to a diabetic patient demonstrated
a potential
relationship
between
impaired
glucose utilization
and
manganese
(109). The patient’s
hyperglycemia
was
corrected
by injection
of manganese
chloride.
This
particular
diabetic patient was resistant
to insulin
therapy.
Another significant
finding was the small
dose of manganese
(20 ,zg) used to produce the hypoglycemic
effect.
Because
the
therapeutic
manganese
dosage used seemed to be a physiological,
rather than
a pharmacological,
amount, the hypoglycemic
effect
was attributed
to the administered
manganese.
In a
recent study the experimental
animal was the sand
rat (Psammomys
obesus),
whose natural diet is high
in manganese.
This animal becomes diabetic when
fed the usual laboratory-animal
commercially
supplied diet and is refractory
to administered
insulin,
but it regains a normal state of health when returned
to its natural,
high-manganese
diet (120). Decreased
concentrations
of manganese
in blood and tissues of
untreated
humans with diabetes mellitus and in pancreatectomized
dogs are consistent
with an effect of
manganese
on blood sugar. A diabetic-like
glucosetolerance
test was observed in manganese-deficient
guinea pigs (38). Dietary supplementation
with manganese completely
removed the abnormality.
A potential relationship
between insulin and manganese
remains to be explored.
Existing evidence is insufficient
to permit speculation on the relation between the hypoglycemic
effect
of manganese
and gluconeogenesis.
Glucocorticoids
exert a permissive action required for the activation
of gluconeogenesis
by physiological
amounts
of cate-
cholamines or glucagon. Adrenal steroids also depress
hepatic uptake of manganese
(137). The effect of the
various hormones
on gluconeogenesis
has not been
studied in manganese-deficient
animals.
Serum
manganese
values
in pathological
states.
Normal serum manganese
values are not firmly established but reportedly range from 1 to 200 gig/liter.
Serum manganese
concentrations
in patients
with
rheumatoid
arthritis,
or infectious,
degenerative,
or
neoplastic
disease are normal, but are decreased
in
diabetes mellitus and are significantly
higher in patients with myocardial
infarction
and massive pulmonary injury. Indeed,
supranormal
serum manganese
concentrations
have been proposed as an index of the
extent and severity of cardiac damage (137). In view
of the difficulties inherent in assaying serum manganese, such a suggestion is now impractical.
Patients
with atherosclerosis
had increased
concentrations
of manganese
in their plasma
and decreased concentrations
in liver, myocardium,
adrenal
glands, pancreas, and kidney (141). In contrast to
atherosclerosis,
the hepatic
content
of manganese
was significantly
increased in hemochromatosis
(3).
The mechanisms
by which tissue manganese
concentrations
vary, as well as the significance
of these variations, are unknown
at present.
No doubt the paucity
of information
on manganese
ease is a function
in manganese
insensitivity
of the inherent
determinations
of most
concentrations
difficulties
in disinvolved
as well as the relative
methods.
Manganese Toxicity
In contrast to deficiency, chronic manganese toxicity in man has been extensively studied. Manganese
is one of the least toxic of the trace metals (137)
and
toxicity occurs only after long, continuous
inhalation
of large quantities
of the element. One reason why
animals are able to tolerate large intakes of manganese may be due to the efficiency of their homeostatic control mechanisms.
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
511
I
Cotzias et al. (23, 85) reported
observations
on
manganese
toxicity in Chilean miners. Symptoms
of
chronic poisoning in manganese
ore miners are seen
after exposures
ranging from seven months to 20
years and sometimes are not seen at all. The clinical
manifestations
of the disease are both psychiatric
and neurologic. The severe psychiatric
symptoms are
often referred to as “locure manganica”
by the local
Chilean populace. The disease progresses to a permanent crippling neurological
disorder of the extrapyramidal system and is, in some ways, clinically similar
to Parkinson’s
disease and Wilson’s disease. In general, clinical disorders
of metal toxicity are caused
mainly by the accumulation
of particular
metals,
such as copper in Wilson’s disease and lead in lead
encephalopathy.
One striking difference seen in manganese toxicity is that the chronically
poisoned patients had a lower tissue manganese concentration,
as
well as a slower turnover
rate, of MMn than did
healthy miners. Chronic poisoning does not necessarily result in elevated tissue manganese concentration;
consequently,
chelation therapy does not seem to be
indicated as it is in the treatment
of other metal poisonings. It is interesting
to note that termination
of
exposure
restored
tissue manganese
concentrations
to normal but the neurological symptoms persisted.
Levodopa
13-(3,4-dihydroxyphenyl)-L-alaninel
is
of known efficacy in the treatment
of Parkinsonism
and is sometimes successful in alleviating manifestations of manganese
intoxication
(20, 24). The rationale for dopa therapy was developed from postmortem studies on patients
with Parkinsonism,
which
showed that dopamine was markedly depleted in the
basal ganglia and substantia
nigra. Because dopamine does not cross the blood-brain
barrier, its precursor, dopa, is used, which does cross the bloodbrain barrier and is presumably
converted
to dopamine in the basal ganglia. Therefore, administration
of dopa effectively reverses the dopamine deficiency
in the Parkinsonian
syndrome (24). The use of dopa
represents
one of the most outstanding
therapeutic
developments
in the treatment
of neurological
diseases. Because the syndrome
of manganese
toxicity
and Parkinsonism
are clinically
indistinguishable,
the same therapy
has been applied empirically
to
both illnesses.
Disappearance
of rigidity and improvement
of postural reflexes, hypokinesia,
and dystonia have been observed
in patients
with chronic
manganese poisoning after therapy with dopa (84).
As mentioned
earlier, mice with the mutant gene
“pallid”
have congenital
ataxia, which can be prevented with large oral doses of manganese. Cotzias et
al. (25) studied these mice as a model of the fundamental relationship
between manganese and biogenic
amines in the brain. Mutant mice had a significantly
slower loss of MMn from the body after intraperitoneal injection of the isotope. These data suggested
that the animals were manganese
deficient. Tissue
manganese
content was measured by neutron activation in mutant and control mice, and these analyses
512
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
indicated that mutant mice have a deficiency of manganese in the brain and in bone. The manganese content of their liver and kidney is normal. Additionally,
L-dopa was administered
orally or intraperitoneally
to mutant and control mice. After oral administration, mutants
showed no cerebral symptoms
compared to the presence of cerebral symptoms
in all
control animals. After intraperitoneal
adminstration
of L-dopa, mutants
had significantly
fewer cerebral
symptoms
than did control mice. Direct analysis
demonstrated
that L-dopa had increased the concentrations of both dopa and dopamine in brain to a significantly
lesser degree in mutant
than in control
mice. Conversion
of intraperitoneally
administered
L-tryptophan
to brain serotonin was also impaired in
the mutant mice.
These data clearly indicate a defect in this animal’s
metabolic capability in handling manganese,
L-dopa,
and L-tryptophan
at a specific site, the brain. Elucidation of this metabolic defect, whether it be related
to the blood-brain
barrier, a defect in manganese
transport,
or a decreased
capacity to metabolize
Ldopa and L-tryptophan,
should provide exciting new
insights into our understanding
of manganese and its
role in neurophysiology.
Summary
Even though manganese
is a small integral part of
total nutrition,
the vital role it plays in metabolic
processes cannot be overemphasized.
Recent studies
have shown that dietary manganese
influences
domestic animals and both environmental
and dietary
manganese
can adversely
affect humans. Although
considerable
attention
has been paid to the role of’
manganese
in many biological functions,
its specific
biochemical
action in vivo remains to be explored.
Knowledge
of the intimate
relationship
between
manganese
and various
hormones,
nucleic acids,
therapeutic
applications,
and biochemical hypotheses
are just beginning
to emerge. The most striking
symptoms
of manganese
deficiency in animals have
been observed
in the offspring of manganese-deficient mothers. Studies on manganese deficiency have
not been done in humans bearing children with congenital anomalies or with mucopolysaccharidoses.
Copper
A considerable
amount of information
on copper is
available
pertaining
to its absorption,
biochemical
role within the cell, and excretion by various species.
As with zinc and manganese,
however, there are also
great voids in our knowledge of the physiology and
biochemistry
of copper.
Absorption.
An adult 70-kg human body contains
80 to 120 mg of copper. The daily copper requirement
for humans has been estimated
to be 2.5 mg per day.
The exact daily consumption
varies greatly from one
geographic
area to another.
The copper content of
grains varies with that of the soil where they are
grown. Copper content of grain determines
the cop-
per content of diets fed to cattle and sheep. Among
foods, shellfish, oysters, organ meats, legumes, dried
vegetables,
and cocoa
contain
relatively
large
amounts of copper. The amount of copper in drinking water depends on its source, the containers used
for storage, and the presence or absence of copper
plumbing.
Copper is absorbed from the stomach and upper
gut by at least two mechanisms.
One, an energy dependent
process, is facilitated
by amino acids and
probably represents
the absorption
of copper complexes of amino acids. A larger portion of the absorbed copper is absorbed by the second mechanism
and is bound to two protein fractions in the intestinal
mucosa. One of these proteins is apparently
the copper enzyme, superoxide dismutase (EC 1.15.1.1). The
second protein involved in copper absorption
is rich
in sulfhydryl
groups and has the characteristics
of
metallothionein.
This protein may act to provide
binding sites for copper which is then slowly released
into the plasma transport
mechanisms
(35). Various
factors within the intestinal lumen or within the gut
mucosal cell seem to play a significant
role in influencing
copper absorption.
Transition
elements
such as cadmium, mercury, silver, and zinc compete
for binding sites on the intestinal mucosa or on the
metallothionein
protein.
Other dietary
ingredients
also influence
copper absorption,
particularly
the
presence
of molybdenum
and the sulfate radical,
which alter or interfere with copper absorption.
The
mechanism
of this antagonism
is unknown.
Co-ingested plant protein diminishes
copper absorption,
perhaps
because
nonabsorbable
complexes
are
formed with phytic acid. Sulfide anions also react
with copper in the gut to form cupric sulfide, which is
insoluble.
This reaction
is used therapeutically
in
Wilson’s disease to diminish
the absorption
of dietary copper.
The actual mechanisms
for copper transport
from
the gut lumen to the blood are unknown.
Copper
in
blood,
and
its
hepatic
distribution.
Copper in the blood occurs in erythrocytes,
bound to
albumin,
and complexed
with ceruloplasmin
and
amino acids. The mean value for erythrocytes
is 980
ag/liter, for plasma 1.09 mg/liter, about 90% of which
is bound to ceruloplasmin.
The total copper content
of erythrocytes
tends to remain constant despite deficiencies of dietary copper or increases in total plasma or hepatic copper. The major portion of copper in
the erythrocyte
occurs as the enzyme, superoxide dismutase. This enzyme, which also occurs in brain and
liver, has the unique role of protecting cells from the
injurious effects of the superoxide
radical, which inhibits cytochrome
c oxidase
(EC 1.9.3.1) (80) in the
electron transport
system. A second component
of
erythrocyte
copper is complexed
with amino acids
and is freely dialyzable.
Plasma albumin appears to be the first carrier to
which absorbed or injected copper is bound. Copper
complexed with albumin rapidly disappears
from the
blood with a concomitant
increase in hepatic copper
content. A small fraction of the total blood copper
appears as amino acid complexes, which are in equilibrium with ionic copper and albumin bound copper.
This amino acid-copper
complex could be important
in the entry of copper into cells and cellular components.
Ceruloplasmin
contains
a major portion
of the
plasma copper in health. This protein is synthesized
in the liver. Radioisotopic
copper is first bound by albumin, then appears in the liver, and finally appears
in ceruloplasmin.
It is of significance that there is no
exchange of ceruloplasmin
copper with nonceruloplasmin copper in the serum. Ceruloplasmin
does not
act as a transport
factor in carrying copper to the
liver from the gut, although it does serve as a means
of distributing
copper to other tissues. The exact
mechanism of this transfer is not known at present.
Excretion.
Very little copper is normally excreted
in the urine, and the source of this urinary copper is
unknown.
Most copper excretion is via the biliary
tract (1), by a process that is quite complex and also
poorly understood.
It is presumed that ionic copper
and copper complexed
to both albumin and amino
acids all contribute to the entrance of copper into the
hepatic cell. Once within the cell, copper is cornplexed with a specific metallothionein.
This intracellular metallothionein
complex is thought to serve as a
temporary
storage form for copper until it enters hepatic lysosomes,
is synthesized
into ceruloplasmin
and copper enzymes, or is excreted
into the bile.
While adequate
knowledge of all the above hepatic
copper complexes is incomplete,
the distribution
of
copper within the hepatic cell and its probable function has been extensively
studied (34). Only 10% of
hepatic cellular copper is found in the microsomal
fraction. Ceruloplasmin
and other copper proteins
are derived
from this fraction. The nuclear fraction
contains about 20% of the cellular copper. There is
evidence that in this fraction copper may react with,
or bind to, polynucleotides.
The significance of these
observations
is not clear. A third subcellular fraction,
consisting
of mitochondria
and lysosomes, contains
20% of the intrahepatic
copper. These lysosomes are
responsible
for much of the copper excreted in the
biliary tract. The remaining hepatic fraction, cytosol,
contains about 50% of the intrahepatic
copper. Here
the primary protein is the sulfhydryl-rich
metallothionein, a protein that is multi-functional:
it releases
copper for enzyme synthesis, ceruloplasmin
synthesis, or biliary excretion. Copper is excreted in the bile
primarily bound to protein macromolecules.
A limited amount of copper is excreted
as copper-amino
acid complexes. The enterohepatic
circulation of copper is minimal
and the macromolecular
copper
species undergoes essentially no reabsorption.
Copper
Anemia.
ubiquity
Deficiency
Copper occurs in sufficient
quantity and
in foods so that the development
of copper
CLINICAL CHEMISTRY, Vol. 21. No.4, 1975
513
deficiency in humans consuming a usual varied diet
and having normal absorption
is unlikely. However,
several instances
of copper-dependent
anemia have
been described
in children (44, 67). In one of these
cases skeletal deformities
occurred that were similar
to those found in animals with copper deficiency
(136).
The similarity
of these human illnesses to
those occurring in other mammals suggests that copper is also an essential element for life, growth, and
development
in humans.
Copper
deficiency
is manifested
in various
ways
among animal species, and such differences
presumably derive from differences in requirements
for various forms of copper compounds
and their resulting
biological activities.
Anemia of varying degree and
type is found in most instances
of copper deficiency
(136) in most mammalian
species. The earliest and
most detailed description
of the anemia was that of
Lahey et a!. in swine (71). This anemia occurred in
association
with low concentrations
of copper in the
blood and was associated with iron deficiency. These
authors also evaluated copper metabolism
in humans
during pregnancy
and in various pathological
states
(72). An earlier study had demonstrated
the presence
of a dark-blue
protein in plasma which contained
copper
and
which
was
named
ceruloplasmin
(55).
These authors also demonstrated
that ceruloplasmin
oxidizes aromatic amines. Although of value diagnostically, the physiologic significance of this reaction is
still uncertain,
although
such metabolically
important substances as epinephrine,
norepinephrine,
serotonin, and melatonin are oxidized in vitro by ceruloplasmin (95). The role of ceruloplasmin
in hematopoiesis has been clarified.
Catalysis of absorbed
or
mobilized iron from the ferrous to the ferric state by
ceruloplasmin
is necessary
for transport
of all iron
used for hemoglobin
synthesis
(35). Although
the
mechanism
is not clear, a neutropenia,
which sometimes precedes the change in erythrocytes,
has been
noted in copper-deficiency
anemia (67).
Enzo#{244}ticneonatal
ataxia.
Incoordination
of movement and gross ataxia are symptoms
found in copper-deficient
neonates
of many species (136). The
pathological
condition seems to be one of demyelination,
producing
a widespread
encephalopathy
with
cavitation and collapse of the cerebrum. Copper concentrations
in the plasma are very subnormal and the
afflicted animals are usually born of mothers grazing
on herbage from copper-depleted
soil.
Various studies correlating
symptoms
and pathological findings with depleted activities of the copper
enzyme, cytochrome
oxidase, indicate strongly that
decreased activity of this enzyme results in the clinical and pathological
findings typical of enzo#{246}tic
neonatal ataxia (56).
The oxidation
of reduced cytochrome
marks the
terminal event in the electron transport
chain. This
reaction
requires
molecular
oxygen and the endproduct is H20. Cytochrome
oxidase catalyzes this
terminal reaction in the electron transport system. It
514
CLINICAL CHEMISTRY, Vol.21,
No.4, 1975
follows that cytochrome
oxidase
is necessary
for all
cellular metabolism.
Cyanide is a lethal agent that
acts with great rapidity because even low concentrations of it can inhibit cyto#{231}hrome
oxidase at low concentrations.
A second factor inherent in the activity
of cytochrome
oxidase is its ability to promote the
formation
of phospholipids
within the central nervous system, which are then incorporated
into myelin
(42). Thus, decreased
copper results in decreased cytochrome
c oxidase
activity,
which results in decreased incorporation
of phospholipid
into myelin.
The primary biochemical abnormality
here may be in
the failure of glycerolphosphate
acyltransferase
(EC
2.3.1.15) and sn-glycerol-3-phosphate
to condense,
which results in an impairment
of phospholipid
synthesis.
Studies in humans have shown only slight variations in serum copper concentrations
in epilepsy (11)
and multiple sclerosis (14). Because a.wide variety of
diseases of the central nervous system are characterized by demyelination,
studies of copper and copper
enzyme activities would seem indicated in these diseases. To our knowledge, such studies have not been
performed.
Defects
in connective
tissue formation.
A defect in
connective tissue formation in copper deficiency has
manifested
itself in many ways. Copper-deficient
chicks produce abnormal
elastic tissue, resulting in
aortic rupture
and malformation
of other vessels
(93). A similar lesion has been described
in copperdeficient swine (144). Hurley has described in detail
the abnormalities
in bone formation-including
gross
malformation,
disorders of epiphyseal
structure,
and
deformities
of joints-associated
with copper deficiency. In all of these abnormalities
the organic matrix, rather than the mineral content, seems to be involved (5).
In a series of elegantly structured
research endeavors it has been shown that monoamine
oxidase (EC
1.4.3.4.), a copper-containing
enzyme, is present in
many animal tissues (54). Abnormalities
in the solubility of elastic tissue have been shown to be associated with, and presumably
due to, low monoamine
oxidase activities.
Monoamine
oxidase catalyzes the
oxidative
deamination
of lysine residues of elastin
peptide chains to produce desmosine, the compound
that forms the cross-links
between elastin fibers in
elastic tissue. The consequent
failure to form normal
elastic tissue results, in turn, in rupture of the aorta
or other large vessels. It seems likely that similar defects in collagen tissue in the bone matrix result from
this common defect. In cattle, copper deficiency results in progressive heart failure characterized
by replacement of normal myocardium
with fibrotic tissue
(9). Sudden
deaths,
presumably
from acute heart
failure, may occur. Abnormalities
of collagen and
connective
tissue related to copper deficiency
have
not been reported in humans. However, to our knowledge, copper studies have not been performed in such
obvious diseases as (e.g.) Marfan’s syndrome, dissect-
ing aneurysm,
Legg-Calv#{233}-Perthes disease (osteochondritis of the capitular epiphysis of the femur), or
idiopathic
myocardial
fibrosis. The role of copper in
these diseases with connective
tissue abnormalities
deserves careful study.
Achromotrichia
and albinism.
Lerner et al. (77)
first demonstrated
that tyrosinase
plays an integral
role in skin pigmentation.
Tyrosinase
(EC 1.14.18.1)
catalyzes the conversion
of tyrosine to 3-(3,4-dihydroxyphenyl)-L-alanine
(dopa). Dopa is subsequently
converted to melanin. A complete lack of tyrosinase
results in albinism in humans. Albinism is characterized by a complete lack of melanin pigment and consequent intolerance
to sunlight. Most animals other
than the pig will also show alteration in pigmentation
of hair or wool when they are copper deficient. The
alterations
in color, texture, and quantity of the wool
of sheep was of economic importance
before the recognition of the adverse effects of copper deficiency.
While the changes in pigmentation
may be derived
from the relative absence of tyrosinase, the failure to
develop or maintain crimp in sheep’s wool appears to
be a result of deficient disulfide groups, which form
cross-linkages
in keratin. This abnormality
may be
similar to the defect in the desmosine cross-linkages
in aortic elastic tissue.
Menkes’
kinky hair syndrome.
originally
described
a progressive
Menkes
et al. (86)
brain disease in in-
fants. This encephalopathy
clinically appeared to involve most of the central nervous system, was accompanied by pili torti, and showed X-linked recessive
inheritance.
Scattered
case reports have added the
additional findings of abnormalities
of the metaphyses of long bone, abnormal
elastic tissue in arteries,
and tortuosity
of cerebral vessels. Danks et al. (30)
reported on seven cases and added hypothermia
to
the clinical picutre described above. He found serum
copper and ceruloplasmin
to be subnormal in his patients. French
[quoted in Evans (35)] stated that
brain tissue in this disease was practically
devoid of
the copper enzyme, cytochrome c oxidase. Danks has
reported some transient
clinical improvement
in his
patients when copper was given parenterally.
Danks
has also shown marked accumulation
of copper in the
intestinal
mucosa of these patients despite low plasma copper concentrations.
Thus, this copper-deficiency syndrome
in humans demonstrated
many of
the abnormalities
described in various animals with
experimental
copper deficiency. The absence of anemia in these infants is remarkable
and is at variance
with the findings in experimental
copper deficiency.
It is readily apparent that the bizarre clinical picture
of copper deficiency in the experimental
animal, long
known to veterinarians
and nutritionists,
has a counterpart
in humans.
Copper
Excess
Wilson’s
disease
tion).
Wilson’s
entity
characterized
(hepato-lenticular
disease
is a well-recognized
by a familial
tendency,
degenera-
clinical
incoordi-
nation, ataxia, progressive mental deterioration,
and
a post-necrotic
hepatic cirrhosis (145). Patients with
Wilson’s disease show decreased
plasma concentrations of copper and of ceruloplasmin.
The relative
amounts of copper bound by amino acids and albumin are increased. The rate at which blood is cleared
of injected copper by all organs, including the liver, is
depressed.
Thus, hepatic uptake of copper is decreased and there is diminished
hepatic synthesis of
ceruloplasmin.
Despite diminished
copper uptake,
tissue copper content is generally increased. The high
concentrations
of copper in liver and brain are of particular importance
because they appear to bear a direct relationship
to the pathologic
dysfunction
displayed by these organs. Urinary copper excretion is
increased but excretion of copper via the biliary tract
is markedly diminished; hence, fecal copper excretion
is decreased.
Extensive
studies by Sternlieb
et al.
(126), Cartwright
et al. (13), and numerous other investigators have produced considerable
evidence that
diminished
excretion of copper through the biliary
tract is the most significant physiological
aberration
involved in copper retention.
Other hypotheses
have
been: (a) increased absorption,
based on higher levels
of radioactivity
in blood
after
administration
of ra-
dioactive copper, (b) increased binding of copper to
hepatic
protein,
and (c) failure
to form
ceruloplasmin.
Although
a diminished
plasma
ceruloplasmin
concentration
is a hallmark of the disease, it
is not the only factor in the pathogenesis
of Wilson’s
disease. Patients have been described
with symptoms
of the disease who have normal or only slightly depressed
concentrations
of ceruloplasmin,
and depressed concentrations
of ceruloplasmin
have been
noted in asymptomatic
persons. Sternlieb et al. (126)
demonstrated
uptake of radioactive copper by the cytosol of hepatic cells in patients with Wilson’s disease. The association
of this copper with small copper-binding
molecules was not significantly
abnormal. Although the rate of transfer of radioactive copper to larger protein molecules of the cytosol and to
the lysosomes was markedly diminished,
the total lysosomal copper content nevertheless
greatly exceeded normal values. Increased
lysosomal copper content seemed to be related to the almost complete lack
of radioactive
copper within the biliary tract. One
modification
of this concept, in regard to the hepatic
binding of copper, may be appropriately
based on the
work of Evans et al. (36). This investigator
continued
his valuable and numerous contributions
to this field
by comparing the binding constant for copper-binding protein (metallothionein)
from patients with Wilson’s disease to that from normal subjects. The apoprotein was prepared in each group and copper metallothionein
was prepared
by equilibrium
dialysis.
The constant for the protein in Wilson’s disease was
four times greater than in the normal, a finding that
lends support to the previous suggestion by Uzman et
al. (138) that the disturbed
balance of copper storage
in Wilson’s disease might be explained by the presCLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
515
ence of an abnormal protein with an increased affinity for binding copper. Walshe (145) has described the
classical manifestations
of Wilson’s disease and has
stressed
that
accumulation
of excess
copper
in the
liver usually precedes that in the central nervous system. Hepatic accumulation
of copper is more marked
and occurs earlier than in the central nervous system.
The mechanism of cellular damage with copper accumulation
is unclear. However, the very interesting
studies by Chvapil have recently been reviewed (16).
These studies show that copper labilizes membranes
and thereby makes cells and organelles more susceptible to injury. Cirrhosis has been produced by injecting copper into experimental
animals.
Biliary obstruction,
particularly,
that found in primary biliary
cirrhosis, results in increased concentrations
of copper and ceruloplasmin
in plasma. The biochemical
abnormality
within the brain substance is also uncertain. However, membrane
lability with excess copper
would seem to be of importance
in the brain as it is in
the case of liver. Copper is deposited in those areas of
the brain that are normally most pigmented,
the subst.antia nigra and the locus caeruleus.
There is no
general agreement
as to anatomical
changes within
the brain.
Treatment
of Wilson’s disease has broadly been directed toward removal of excess body stores of copper. Chelating agents, first BAL (2,3-dimercaptopropanol) and later penicillamine,
have been helpful in
increasing
the urinary excretion of copper. Limitation of copper in the diet and addition of potassium
sulfide, which will diminish copper absorption,
have
also proved helpful. Even with these measures, maintaining a negative copper balance may be difficult.
Early diagnosis, and persistent
vigorous therapy will
usually be rewarded by amelioration
of symptoms or
cessation in the progression of the disease.
Hormonal
factors
in copper
metabolism.
Although
knowledge of the abnormalities
to be found in the
serum copper concentrations
are of great value in detecting patients with Wilson’s disease, these can only
be properly interpreted
when knowledge of other factors affecting
copper metabolism
are appreciated.
First consideration
must be given to the factors producing physiological
alteration
in plasma copper. Important
among these is the effect of hormones
on
copper metabolism.
The pituitary,
through elaboration of growth hormone and its anabolic effect on
protein synthesis, seems to affect copper metabolism.
Hypophysectomized
rats,
examined
three
weeks
after
operation, tend to have a greater hepatic copper content than controls.
Administration
of growth hormone to hypophysectomized
rats resulted in diminished hepatic copper content. The action of corticotropin is unclear except when it causes alteration
of
adrenal cortical secretion. The relation between adrenal cortical function and copper metabolism
is itself
complex. Supranormal
serum copper values are reportedly associated
with decreased
adrenal cortical
activity.
Repeated
experiments
have demonstrated
516
CLINICAL
CHEMISTRY,
Vol. 21, No.4,
1975
that adrenal steroids depress hepatic copper concentrations primarily by enhancing the biliary excretion
of copper (83). In part, this corticosteroid
effect results from the increased
rate of bile secretion that
cortisone induces. A circadian pattern of variation in
serum copper and ceruloplasmin
has been discerned.
Lower concentrations
of copper and ceruloplasmin
occur at times when corticotropin
and hydrocortisone
concentrations
are highest (78, 90). The mechanism
whereby high serum copper occurs in adrenal insufficiency is extremely
complex.
This process is discussed further in the section on zinc. Injections
of
epinephrine
tend to produce increases in plasma copper and ceruloplasmin
in control and adrenalectomized rats, although the mechanism
is not clear and
may be more broadly related to stress situations.
In
particular,
exhausting
tended
by increases
plasmin.
physical
in
plasma
exercise
copper
has been atand cerulo-
Experiments
in various species of mammals
have
resulted in divergent
observations
regarding
the effect of the thyroid gland on copper metabolism.
The most striking effect on copper and ceruloplasmin in the plasma are the increases produced by
estrogens.
These hormones
increase physiologically
during pregnancy
and their effects on copper are
readily reproduced
by the injection or ingestion of estrogens. The effect appears to be the result of estrogenic stimulation
of ceruloplasmin
synthesis.
Serum
ic states.
copper
values
in physiologic
and patholog-
There are several clinical conditions
in humans in which low concentrations
of copper are
found in the serum.
Hypocupremia
occurs with
kwashiorkor,
in which inadequate
copper may be ingested and its absorption
may be impaired secondary
to the protein-calorie
deficit. In sprue and celiac disease copper values are low for similar reasons. They
also are low in nephrosis, and the urinary loss of copper and copper-binding
protein seem to be responsible. Many acute and chronic diseases are accompanied by increased concentrations
of copper and ceruloplasmin in the serum. Often these increases may be
related
to ceruloplasmin
being
an acute
phase
reac-
tant. The effect of leukocyte endogenous
mediator in
increases in acute phase reactant has been discussed
in the section
on zinc.
Increased
concentrations
of ceruloplasmin
and
serum copper occur in pregnancy
and with estrogen
therapy,
as in the use of oral contraceptives.
Extremely high concentrations
of ceruloplasmin
have
been found
in various
lymphomas,
particularly
Hodgkin’s disease. Response to therapy, when successful, may be accompanied
by a return of serum
copper values to normal and re-activation
of the disease may be anticipated
when the serum copper and
ceruloplasmin
values begin to rise.
Copper
Toxicity
Copper toxicity is relatively uncommon. The ingestion of more than 15 mg of elemental copper usually
produces nausea, vomiting, diarrhea,
and intestinal
cramps. In more severe cases, intravascular
hemolysis
is seen. This phenomenon
has also been seen in renal
dialysis units in which an excess of copper was transferred from the dialysis bath to the patient. Studies
of this hemolytic anemia indicate that copper sulfate
may inhibit glucose-6-phosphate
dehydrogenase
(EC
1.1.1.49) activity, inhibit erythrocyte
glycolysis, denature hemoglobin, and oxidize glutathione
(40). Any of
these processes could result in hemolysis. In India,
ingestion of copper sulfate is a relatively
common
way of committing
suicide. Studies by Chuttani et al.
(15) have shown that severe cases develop jaundice,
dilatation
of the central veins of the liver, and varying degrees of acute hepatic necrosis. Tubular swelling, glomerular
congestion,
and hemoglobin
casts in
the urine were frequently found.
Summary
The present knowledge of copper metabolism
and
the disease states resulting from copper deficiency
and excess have been reviewed briefly. Even the wellrecognized pathological
states relating to copper are
not accompanied
by a complete knowledge of the biochemical disturbances
resulting from the copper abnormality.
The precise mechanism
resulting in Wilson’s disease is not established.
Why species differences occur in response to copper deficiency is not
known. The presence
of normal
erythrocytes
in
Menkes’ disease, when all copper stores should be depleted, is intriguing,
and contrasts
with the easily
produced
copper-deficiency
anemia found in pigs.
Rapid continuation
in the growth of knowledge in
this field is certain. It seems apparent
to us that
growth in this area depends on basic research in enzymology, trace-element
interactions,
and the effect
of trace elements on membranes.
Finally, dissemination of this information
to the practicing
physician is
of equal importance
if he is to recognize known disorders in human trace element metabolism
and to discover presently unknown disorders.
Concluding Remarks
In preparing this review it was apparent to us that
advances
in the field of trace elements
have been
made rapidly in a wide variety of disciplines.
We
have tried to summarize
these advances
for zinc,
manganese, and copper, particularly
as these findings
relate to humans. At the same time we have tried to
designate areas where information
is lacking or is incomplete. In a few instances we have attempted
to
speculate
in order to integrate
diverse information
from various fields into a unified thesis.
Of particular
interest to us with regard to zinc,
manganese,
and copper was a recurring theme associated with the deficient state. Deficiency of each of
these elements was associated with characteristic
integumentary
and skeletal abnormalities,
congenital
anomalies (most dramatic in the case of zinc deficiency), defects in growth and development,
and abnor-
malities in sensory perception.
We believe that the whole field of trace-element
metabolism,
as it relates to man, is in the prepubertal
stage of growth and development.
A tremendous
growth phase lies just ahead. Thus, we have tried to
delineate where we stand today in our knowledge of
these three elements and, perhaps to some extent,
where we are going in the future.
We wish to thank the thousands
of investigators
whose work we
have reviewed;
without
them this manuscript
would not have been
possible.
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