CLIN.CHEM.20/9, 1141-1145(1974)
Manganese, Copper, and Zinc Concentrations in
Serum and Packed Blood Cells during Acute Hepatitis,
Chronic Hepatitis, and Posthepatitic Cirrhosis
Jacques Versieck, Fabrice Barbier, Albert Speecke, and Julien Hoste
Manganese, copper, and zinc concentrations
termined
in serum
and packed
were de-
blood cells of normal
controls, patients with acute and chronic (persistent or
aggressive) hepatitis, and cases of postnecrotic cirrhosis. During the active phase of acute hepatitis, serum
manganese concentrations
are invariably increased; the
difterence between the mean value and the normal is
highly significant, P< 0.001. The mean serum copper is
also significantly increased (P < 0.01). The concentra-
tions become normal during the subsiding phase. In
chronic aggressive hepatitis and posthepatitic cirrhosis,
the mean serum manganese
concentration
is increased, P < 0.001, whereas the serum zinc concen-
tration is frequently decreased. There is a highly signif icant (P < 0.001) positive correlation between serum
manganese concentration
and the activity in serum of
aminotransferases,
in subjects with acute or chronic
hepatitis or postnecrotic cirrhosis.
Addftional Keyphrases: trace elements #{149} liver disease
neutron-activation analysis
#{149} aminotransferases
normal values #{149} bilirubin
#{149}
We developed a blood-sampling
technique and an
analytical
method for manganese, copper, and zinc
that decreases contaminations
to a neglible
level.
Some results obtained
in control subjects
were published elsewhere
(1, 2).
Several reports concerning
copper and zinc metabolism in liver disease
have already
been published
(3-12), but we are not aware of data about manganese in serum and packed blood cells.
Here,
we report
manganese,
copper,
and zinc con-
centrations
in serum and packed
blood cells during
the active and subsiding
phase of acute, icteric viral
hepatitis,during chronic persistent and chronic
aggressive hepatitis,
and during posthepatitic
liver cirrhosis.
Department
of Internal
Medicine,
Division of Gastroenterology,
Academic
Hospital,
De Pintelaan
135, B-9000 Ghent; and the Laboratory
for Analytical
Chemistry,
Institute
for Nuclear
Sciences,
Proeftuinstraat
86, B-9000 Ghent [both of the State University
of
Ghent, Belgiumi.
Received
April 29, 1974; accepted
June 10, 1974.
Materials and Methods
Forty-six
amined.
controls
Of
(25 men and 21 women)
these,
ophthalmologic
27
ward,
were
hospitalized
one in the orthopedic
were exin
the
ward,
and one in the internal
medicine
ward. Seventeen
subjects
were studied
during
a clinical examination
for recruitment
of university
personnel.
All were free
of any obvious
medical
disease.
Electrocardiogram,
chest x-ray and laboratory
examinations
(including
sedimentation
serum
rate, erythrocyte
bilirubin,
cholesterol,
and leukocyte
alkaline
count,
phosphatase
(EC 3.1.3.1), thymol turbidity,
aspartate and alanine
aminotransferases
(EC 2.6.1.1 and 2.6.1.2), serum
total protein, albumin, and globulin) were normal.
Subjects taking oral contraceptives
were not included
as it is known that these drugs influence copper and
zinc concentrations
in serum (6, 8, 13-16).
The patients with hepatic disease were hospitalized in the gastroenterology
clinic. The diagnosis of
acute icteric viral hepatitis was based on clinical and
biochemical
evidence.
In one case the diagnosis
of
acute cholestatic
viral hepatitis
was made. There was
one case of acute fulminant
liver failure resulting
in
fatal hepatic coma. In four subjects the samples were
taken during the active phase (17) of acute hepatitis,
in four others during the subsiding
phase (17), and in
six patients both during the active and the subsiding
phase. In all cases of chronic hepatitis
the diagnosis
was proved by peritoneoscopy
and liver biopsy. Cases
of chronic hepatitiswere divided into a chronic per-
sistent
and
a chronic
aggressive
form according
to
histological
criteria
(18, 19). One case of chronic aggressive hepatitis
was studied
shortly after she recovered from a hepatic
coma provoked
by gastrointestinal bleeding.
The diagnosis
of posthepatitic
cirrhosis
(five patients)
was based on a documented
clinical
history and on biochemical,
peritoneoscopic,
and histological evidence.
Liver-function
tests were done by standard
techniques (20). Hepatitis
B antigen
(HB Ag) was determined by counterelectrophoresis
or by the comple-
CLINICALCHEMISTRY,
Vol. 20. No.9, 1974 1141
ment fixation reaction.
Of the 10 cases examined
during the active phase
of acute hepatitis,
four were HB Ag positive. The
four cases with chronic persistent
hepatitis were HB
Ag negative. Two of the four cases with chronic aggressive hepatitis and four of the five patients with
postnecrotic
cirrhosis were HB Ag positive.
Manganese,
copper, and zinc concentrations
in
serum and packed blood cells were determined
by
neutron-activation
analysis. The collection of samples, the irradiation,
chemical separations,
and measurements
were all done as described elsewhere (1).
However, in most cases of the present study, blood
was collected
with a plastic
cannula
trocar
(Intranule
110 16; Vygon Sterile,
Ecouen, France) instead of a
disposable
steel needle (18 G 14. Terumo). As proven
by contamination
studies according to a technique
published previously (21) this method appeared to be
preferable
for the collection of the blood samples.
Collection of blood was performed between 8 and 10
a.m. after an overnight fast.
Utmost
care was taken
to avoid
all possible
sources
between the manganese concentration
in packed cells
is slightly significant
(0.02 <P < 0.05) and between
the zinc concentration
in serum it is significant (0.001
<P < 0.01). This last statement
agrees with the findings of Lindeman
et al. (25).
The mean values and standard deviations of serum
manganese, copper, and zinc, determined
in patients
with acute hepatitis, chronic hepatitis, and posthepatitic cirrhosis, are also summarized
in Table 1.
During the active phase of acute icteric viral hepatitis, we always observed an increased serum manganese concentration.
The mean value is 2.32 ng/ml,
highly significantly
different from the normal values
(P < 0.001). The highest value (4.09 ng/ml)
was
found in the patient with fulminant
hepatic failure.
There also is a slight increase of the mean serum copper concentration
(P < 0.01), the mean value being
1.43 g/ml and the highest 2.22 g/ml (in the case of
cholestatic viral hepatitis). The mean serum zinc concentration does not change significantly
(mean ± SD:
0.90
± 0.16 tg/ml).
appears
One
to be lowered
serum
zinc value,
however,
(0.59 ig/ml).
of contamination.
Manipulations
were carried out in
a dust-free room. The samples were collected and irradiated in containers cleaned with extreme care (1).
During
the subsiding
phase of acute icteric viral
hepatitis,
the serum manganese
concentrations
normalize (mean ± SD: 0.60 ± 0.12 ng/ml). In one case of
It should
positive HB Ag + hepatitis, four months after a mitral valve prosthesis,
the serum copper appeared to
be markedly increased
(2.32 zg/ml).
The serum zinc
concentration
is normal in all cases (mean ± SD: 0.91
± 0.07 g/ml).
be noted
that
packed
cells were
irradiated
at a reactor site with a thermal- to fast-neutron
flux
ratio of 90, as the iron content is very high. This decreases the interference
caused by the 56Fe (n.p.)
56Mn reaction to about 7%. We applied a correction
for this error. All samples were analyzed in duplicate.
The percent standard
error for the method is approximately
8% for manganese,
3% for copper, and
7% for zinc.
Means
using
and standard
standard
subjects
deviations were calculated by
(22). The results for normal
for outlying values by the meth-
equations
were tested
ods of Grubbs
(23). The normality
of the distributions
in normal
subjects
was checked
by the chisquare test (24). For the comparison
of two means,
The paired values of serum manganese
determined
in six patients
during the active and later during the
subsiding
phase of acute icteric viral hepatitis
are
shown in Figure 1. The difference
of the two means is
highly significant
(t = 4.885; 0.001 <P
< 0.01). For
serum copper and zinc, no significant
difference
was
found
between
the active and subsiding
phase
in
these six patients
(serum copper, t = 1.230; serum
zinc, t = 1.145).
the usual equations
were used for paired or for unpaired cases (22). The regression line and the correlation coefficients (r) between serum aminotransferases and serum manganese values were calculated after
logarithmic
transformation
of both
variables
3.0
(22).
2.5
Results
2.0
The mean values, standard deviations, and ranges
for manganese, copper, and zinc in serum and packed
blood cells of normal subjects are set forth in Table 1.
All distributions
are normal, but the following values
are outlying:
1.01 ng/ml (males) and 1.04 ng/ml (females)
(serum manganese),
1.99 tg/ml
(females)
(serum copper), 36.9 ng/g (females)
(packed
cells
manganese), 0.96 .tg/g (females) (packed cells copper)
and 15.5 tg/g (females) (packed cells zinc).
No difference of copper and zinc concentration
in
packed
cells is found between
men and women
whereas the difference for manganese and copper in
serum is not statistically
significant.
The difference
1142
CLINICAL
CHEMISTRY,
Vol. 20, No. 9, 1974
tS
1.0
C
0.5
“S”
Actve
phase
-
Subsdinq
phase
Acuteictericviralhepatitis
Fig. 1. Values for serum manganese determined in six
subjects during the active and during the subsiding phase of
acuteicteric
viral
hepatitis
Table 1. Values of Manganese, Copper, and Zinc in Normal Controls and in Patients with
Acute and Chronic Hepatitis and Posthepatitic Cirrhosis
Packed
Serum
Mn
ClassifIcatIon
(and
number)
Males(25)
Range
Females (21)
Range
Overall (46)
Range
Cu
Zn
Mn
ng/mI
pgfml
pg/mi
0.59
±0.12
0.45-1.01
0.55
±0.14
0.38- 1.04
0.57
±0.13
0.38- 1.04
1.03
±0.18
0.73- 1.51
1.13
±0.29
0.77 -1.99
1.07
±0.24
0.73-1.99
0.99
±0.13
0.72- 1.21
0.88
±0.11
0.69- 1.15
0.94
±0.13
0.69- 1.21
Acuteviral
hepatitis
Active phase (10)
Acute viral hepatitis
Subsiding phase (10)
Chronic persistent
hepatitis (4)
Chronic aggressive
hepatitis (4)
Postnecrotic cirrhosis
(5)
ng/g
13.6
±3.2
8.1
20.5
16.9
±6.0
9.3- 36.9
15.0
±4.9
8.1
36.9
-
-
2.32
1.43
0.90
18.8
±0.96
0.60
±0.36
1.27
±0.16
0.91
±9.2
18.9
±0.12
0.58
±0.13
±0.41
±0.17
±0.07
1.02
±0.16
±10.3
13.7
±2.4
0.84
±0.24
0.84
1.25
±0.31
1.09
0.79
±0.20
0.81
14.8
±4.0
18.2
±0.20
±0.30
±0.33
1.04
blood Cells
Cu
Zn
pg/ge
pg/ge
0.66
±0.06
0.54-
0.80
0.66
±0.10
0.53-0.96
0.66
±0.08
0.53- 0.96
10.1
±1.1
7.8
11.6
10.1
±1.8
6.7
15.5
10.1
±1.4
6.7- 15.5
-
-
0.66
±10.2
9.9
±0.08
±1.7
0.66
±0.05
9.5
±1.0
0.65
±0.07
0.66
±0.07
0.62
10.0
±0.3
10.2
±1.0
10.3
±0.07
±1.7
Values
are themean ±1 SD. Many ofthese distributions
areskewed.
Pergram wet wt.
10
In the four cases of chronic persistent
hepatitis,
serum manganese, copper, and zinc remain at normal
values (Table 1).
In cases of chronic aggressive hepatitis,
the mean
serum manganese
concentration
(0.84 ng/ml) is significantly
(P < 0.00 1) higher than the mean of the
controls,
the mean
serum
copper
concentration
(1.25
Lg/ml) does not significantly
differ from normal and
the mean serum zinc concentration
(0.79 ig/ml) is
significantly
(0.02 <P <0.05) decreased.
In cases of postnecrotic
cirrhosis, the mean serum
manganese content (0.84 ng/ml) is significantly
(P <
0.001) increased. The mean serum copper concentration (1.09 g/ml)
is normal. The mean of the serum
zinc concentration
(0.81 tg/ml) does not significantly
differ (0.05 <P < 0.10) from the mean of the normal
controls. However, in three cases of advanced cirrhosis, the
serum
zinc
concentration
was definitely
bilirubin
and serum
manganese
0.1
0.1
/,
“ 10
100
Aspartate
10000
1000
amiflotraflSferaS(
(Karmu,its)
Fig. 2. Individual values for serum manganese and aspartate
aminotransferase activity in patients with acute and chronic
hepatitis and postnecrotic cirrhosis, plotted in a double-log
scale diagram. r = 0.945
low
(respectively,
0.62 tg/ml, 0.47 tg/ml, and 0.65 sg/ml).
There is a significant positive correlation
between
serum
c
concentrations
in acute and chronic hepatitis and postnecrotic
cirrhosis (r = +0.43; 0.01 <P < 0.02). There also is a
highly significant (P < 0.001) positive correlation between serum aminotransferase
activities and serum
manganese concentrations.
If plotted in a double logarithmic diagram, the relationship
is linear (Figure
2). The correlation coefficient calculated after double
logarithmic
transformation
is +0.945 and +0.870 for
aspartate
aminotransferase
and alanine aminotrans-
ferase,respectively,versus serum
manganese.
The
equation of the regression line is y = 0.434 x
0.973,
y being the log of serum manganese (in ng/ml) and x
the log of aspartate
aminotransferase
(in Karmen
units).
Manganese,
copper, and zinc concentrations
in
packed blood cells do not show significant variations.
The results are summarized
in Table 1. One exceptionally high value for manganese in packed cells was
found during the active and subsiding phase in a 36year-old woman with post-transfusion
hepatitis
(respectively, 39.6 and 45.2 ng/g).
-
CLINICALCHEMISTRY, Vol.20,No. 9, 1974
1143
Discussion
Most of the reported manganese,
copper, and zinc
concentrations
in packed blood cells (1, 3, 25-30) and
copper and zinc concentrations
in serum from normal
subjects (1, 4-9, 13-16, 25-29, 31-44) show substantial agreement.
Until recently, however, confusion
has persisted concerning
the normal serum manganese concentration
(1, 29, 34, 44-52).
Mean values
published
during the last decennium
vary between
0.587 (45) and 24 ng/ml (51). It appears that the
mean serum manganese
value is very low and that
the values in 46 control subjects approach a normal
distribution
with a quite small dispersion
(0.57 ±
0.13 ng/ml) (2). The upper limit was found to be 1.04
ng/ml. Higher values described
in normal subjects
are apparently
due to contaminations.
The values reported in the present study for manganese in packed
blood cells and for copper and zinc in packed blood
cells and serum agree with other recent data (4, 7, 15,
25, 26, 29, 33, 37, 38). The concentration
of manganese in packed blood cells is about 26-fold higher
than in serum.
Our study shows that an increased serum manganese concentration
is a constant finding during the
active phase of acute viral hepatitis. A slight but statistically significant increase in the mean serum copper concentration
was also observed, whereas the zinc
concentration
remained within normal limits except
in one case with a low value. During the subsiding
phase, normalization
of the manganese
and copper
serum concentrations
is observed.
We suggest that two hypotheses can be postulated
to explain the invariably increased serum manganese
during the active phase of acute viral hepatitis: necrosis of liver parenchyma,
leading to an important
release of hepatic manganese, or decreased hepatobiliary excretion. Indeed, the manganese content of the
liver is very high as compared with its concentration
in serum (1.5 ± 0.2 Lg/g of wet liver (53) versus 0.57
± 0.13 ng/ml of serum), and it has been demonstrated that the liver has an important role in the excretion of manganese
(54, 55). Comparison
of the
liver copper content with serum concentrations
also
shows a difference (7 sg (56) or 11.6 ± 4 g/g of wet
liver (57) versus 1.16 ± 0.37 zg/ml of serum). Copper
is also principally
excreted into the bile (56). Thus,
similar mechanisms
can be postulated
for the increase in serum copper during acute hepatitis. There
is no full agreement concerning the plasma or serum
zinc concentration
during acute hepatitis (4, 7, 10, 11,
58). Our results agree with previous findings of some
authors (4, 58) but disagree with those of others (7,
10, 11). The normal zinc concentration
in liver is reported as being 67 ± 20 sg (56) or 70 tg/g of wet liver
(27). During acute icteric viral hepatitis,
however, an
increased urinary zinc excretion has been reported (7,
11). It seems possible that the release of zinc during
liver cell necrosis is counterbalanced
by an increased
urinary zinc excretion, resulting in a normal serum
zinc value in most such patients.
1144
CLINICAL
CHEMISTRY,
Vol. 20, No. 9,
1974
In our cases of chronic persistent
hepatitis, no abnormalities
of serum manganese, copper, or zinc concentration were observed.
In cases of chronic aggressive hepatitis and postnecrotic
cirrhosis,
a statistically
significantly
increased serum manganese
level was also found. The
values for serum zinc are dispersed. Abnormally
low
values were found in one case of chronic aggressive
hepatitis
and in three cases of advanced
cirrhosis.
These observations
are in harmony with previously
reported data (10).
We believe that the statistically
highly significant
positive correlation
between serum aminotransferases and serum manganese permits us to postulate that
an increased value of the latter can be an index to
liver cell damage.
Although some manganese can be excreted by auxiliary gastrointestinal
routes, especially in conditions
of overloading,
excretion by way of the bile constitutes the main regulatory route (54). Thus, determinations of serum manganese
concentrations
in various cholestatic syndromes are also needed.
We would like to acknowledge
assistance
in statistical
analysis
from Miss S. De Bie, technical
assistance
of Misses H. Michels and
L. Vanballenberghe
and Messrs. J. De Rudder
and G. Piens, and
the secretarial
assistance
of Mrs. Y. Odent.
This work was supported
by the National
Fonds voor Wetenschappelijk
Onderzoek
and the Interuniversitair
Instituut
voor
Kernwetenschappen.
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No. 9,
1974
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