Download Zinc supplementation and amino acid

Zinc Supplementation and Amino Acid–Nitrogen
Metabolism in Patients With Advanced Cirrhosis
GIULIO MARCHESINI, ANDREA FABBRI, GIAMPAOLO BIANCHI, MARA BRIZI,
Zinc deficiency is common in cirrhosis and has been
involved in the altered nitrogen metabolism. In this
study, we measured the effects of zinc supplementation
on the dynamics of amino acid–derived urea synthesis
in cirrhosis with mild or latent encephalopathy. The hepatic conversion of amino acids into urea was studied in
eight patients with advanced cirrhosis under controled
conditions of substrate availability (continuous alanine
infusion), before and after 3-month oral zinc sulfate supplementation (600 mg/d). Eight more patients, matched
for hepatocellular failure and encephalopathy, served
as controls. Plasma zinc levels were reduced in all patients and returned to normal after oral zinc. The alanine-stimulated urea nitrogen synthesis rate in relation
to a-amino-N concentration—the functional hepatic nitrogen clearance—increased by 25% after zinc supplementation, i.e., more urea was produced at any a-aminoN concentration. Basal and alanine-induced glucagon
decreased by 50%, and the ammonia response to alanine
decreased by 30%. Psychometric tests improved, as did
routine and dynamic liver function tests and the ChildPugh score. Also, the plasma concentration of lipid peroxides was reduced by zinc. No significant changes were
observed in the control group. Our data indicate that
long-term oral zinc speeds up the kinetics of urea formation from amino acids and ammonia. Changes in the hormonal drive and/or the antioxidant activity of zinc might
be involved in the general improvement in liver function, whereas the beneficial effects on encephalopathy
might stem from decreased ammonia. (HEPATOLOGY
1996;23:1084-1092.)
Zinc is considered an essential trace element for several metabolic processes, exerting a protective action
on liver cell activity and possibly preventing cellular
damage caused by oxidative stress.1 Reduced zinc conAbbreviations: OTC, ornithine transcarbamoylase; FHNC, functional hepatic nitrogen clearance; NCT, number connection test; CRTs, continuous reaction times to sound; UNSR, urea-N synthesis rate; TBW, total body water;
GEC, galactose elimination capacity; TBARS, thiobarbituric acid reacting substances.
From the Istituto di Clinica Medica Generale and Cattedra di Malattie del
Metabolismo, Università di Bologna, Policlinico S. Orsola, Bologna, Italy.
Received March 2, 1995; accepted December 11, 1995.
Supported by a grant from Ministero dell’Università e della Ricerca Scientifica, Fondi 40%, Rome, Italy.
Address reprint requests to: Giulio Marchesini, M.D., Istituto di Clinica
Medica Generale e Terapia, Università di Bologna, Policlinico S. Orsola, 9, Via
Massarenti, I-40138 Bologna, Italy.
Copyright q 1996 by the American Association for the Study of Liver
Diseases.
0270-9139/96/2305-0023$3.00/0
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MARCO ZOLI
tent is common in patients with advanced cirrhosis,
particularly of alcohol origin,2 but the biochemical basis
for zinc deficiency is still unknown. Several factors,
such as poor dietary intake, impaired intestinal absorption, and excessive urinary losses may be responsible
for reduced whole-body zinc content.3
The importance of zinc deficiency in precipitating episodes of hepatic encephalopathy is a matter of discussion. In a single patient with cirrhosis and severe recurrent hepatic encephalopathy, zinc levels after zinc
supplementation and artificially induced zinc deficiency correlated closely with mental state and electroencephalography tracings.4 In a randomized doubleblind trial, zinc sulfate oral supplements increased to
normal plasma zinc levels of cirrhotic patients and significantly improved mild encephalopathy of the chronic
type.5 During treatment, ammonia levels decreased,
and plasma urea concentration increased. The results
were not confirmed in a short-term crossover study
with zinc acetate supplements, which failed to normalize plasma zinc levels.6 Also episodes of acute encephalopathy after gastrointestinal hemorrhage have been
successfully treated with zinc.7 In cirrhotic rats, zinc
supplementation was shown to increase the hepatic activity of ornithine transcarbamoylase, a key-enzyme of
urea cycle.8 This was accompanied by increased urea
formation and decreased ammonia levels, which might
be the biochemical basis for the beneficial effects of zinc
on mental state in humans.
The liver plays a pivotal role in amino acid/protein
disposition. Most of the amino acid nitrogen that is not
used for protein synthesis is converted by hepatocytes
into urea, which is irreversibly lost in the urine. The
process may be quantified, after standardization for
substrate availability, by the slope of the regression
of urea-nitrogen synthesis rate during defined timeintervals on the corresponding average a-amino-nitrogen concentrations, the so-called functional hepatic nitrogen clearance (FHNC).9 The technique proved useful
to study the effects of disease, hormone, drugs, and
dietary manipulations on the dynamics of amino acid–
derived urea synthesis.9
In the present study, we assessed the effects of 3month oral supplements of zinc sulfate on the hepatic
conversion of alanine nitrogen into urea nitrogen in a
group of patients with advanced cirrhosis, under controled conditions of substrate availability induced by
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TABLE 1. Clinical and Laboratory Data at the Beginning of the Observation
Period in the Two Groups of Patients With Cirrhosis
Case No.
Age
(yr)
Experimental group
1
43
2
68
3
50
4
61
5
39
6
46
7
59
8
43
Mean (SD)
Control group
9
47
10
57
11
60
12
61
13
46
14
53
15
54
16
47
Mean (SD)
Normal values
Cause of
Cirrhosis
Albumin
(g/L)
Prothrombin
Activity (%)
Zinc
(mg/dL)
ChildPugh Score
Ammonia
(mmol/L)
NCT
(sec)
Abnormal
CRTs (%)*
HCV
Alcohol
HCV
HCV
Alcohol
Alcohol
HCV
Alcohol
42
33
22
41
41
34
25
34
34 (8)
60
75
35
58
68
47
60
51
58 (13)
80
53
53
55
83
73
84
63
68 (14)
6
8
13
8
6
8
10
9
8.5 (2.3)
17
55
53
38
45
30
18
61
42 (17)
52
50
170
80
49
80
120
60
84 (42)
13
44
21
29
39
29
26
10
26 (12)
Alcohol
HBV
Alcohol
HCV
Alcohol
HCV
HCV
HCV
28
24
42
27
36
25
30
38
31 (7)
ú4.0
60
44
52
50
65
47
58
68
56 (9)
ú80
80
54
53
55
63
64
63
84
65 (12)
ú80
9
10
8
10
7
11
8
6
8.6 (1.7)
—
55
86
38
18
30
38
70
22
45 (24)
õ35
92
110
80
106
52
83
107
52
85 (24)
õ50
36
36
28
34
10
42
43
18
31 (10)
õ15
Abbreviation: HCV, hepatitis C virus; HBV, hepatitis B virus.
* Number of CRTs ú400 msec in a series of 100.
continuous amino acid infusion. A second group of patients, with similar hepatocellular failure and encephalopathy, prospectively followed without any dietary intervention, served as controls. The results show that
zinc sulfate supplementation increases the rate of urea
synthesis, reduces plasma ammonia in response to an
amino acid load, and, finally, improves mild or latent
encephalopathy.
PATIENTS AND METHODS
Subjects. Two groups of eight patients with hystologically
documented cirrhosis and stable clinical conditions were
studied. The first group (experimental group) was composed
of seven men and one woman, 39- to 68-years-old (median,
50 years), with cirrhosis of alcoholic (four cases) or hepatitis
C virus origin (four cases). Their clinical and laboratory data
are reported in Table 1. Three subjects were in fairly good
nutritional conditions, whereas the remaining five had clinical evidence of reduced lean body mass. Two patients were
in Child-Pugh class A,10 four cases were in class B, two were
in class C. Four patients had episodes of variceal bleeding at
least 2 months before the study. Four patients had mild ascites at ultrasonography, which was not clinically evident, and
all were being treated with diuretics (spironolactone [100200 mg/d] and/or furosemide [25 mg/d]) and lactulose (15-30
g/d). Clinical evidence of chronic hepatic encephalopathy was,
nonetheless, present in two patients (patients 3 and 7),
whereas the remaining six patients had latent encephalopathy, expressed by alterations in psychometric testing (number-connection test [NCT]11 and continuous reaction times to
sound [CRTs],)12 or fasting hyperammonemia (Table 1). Two
of these last patients, in Child-Pugh class A at the time of
study, had shown clinical signs of encephalopathy in the last
6 months.
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Patients of the second group (control group) were seven
men and 1 woman, aged 46 to 61 years (median, 55 years),
with cirrhosis of viral (five cases) or alcoholic origin (three
cases). Their clinical and laboratory data were well matched
with those of patients in the experimental group (Table 1).
One patient was in Child-Pugh class A, five were in class B,
and two were in class C. These patients also had signs of
overt (patients 10, 12, and 15) or latent encephalopathy at
the time of study and were being treated with lactulose. In
addition, all were receiving diuretic treatment for previous
episodes of ascites.
Patients with alcoholic cirrhosis had been abstaining from
alcohol for at least 1 year before the study, and two patients
had stopped drinking alcohol 2 years before. Renal function
was normal (plasma creatinine, õ1.3 mg/dL), and there was
no evidence of previous or actual endocrine diseases and/or
complicating disorders at the time of study. During the study
all patients were on a standard hospital diet to provide 30 to
35 kcal and 0.8 g protein/kg body weight.
After basal assessment, patients in the experimental group
received an oral supplementation of zinc sulfate (200 mg
three times a day for 3 months), prepared by the pharmaceutical department of our hospital. All other medications (diuretics and lactulose) were continued unchanged throughout
the study period. Patients were regularly followed as outpatients (every month), and compliance to zinc treatment was
checked by counting the number of tablets not used in the
previous 30 days. In one patient, who complained of gastrointestinal symptoms after zinc treatment, the final evaluation
was anticipated by 15 days. The control group also was prospectively followed for 3 months, without any additional dietary intervention. During the study period, no patient in
either group had episodes of acute encephalopathy, and none
received unabsorbable antibiotics. None of the patients in the
experimental group showed signs of zinc toxicity.3
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In two patients from the experimental group, all tests were
repeated a third time, approximately 6 months after the end
of zinc sulfate treatment, to study the effects of zinc withdrawal. This time period was considered to account for a
possible carry-over effect of zinc supplementation.
All subjects gave informed consent to take part in the
study. The protocol was submitted to and approved by the
Ethical Committee for Human studies operating in our department.
Methods. All experiments were performed in the course of
hospital admission, at the beginning and at the end of the 3month study period. Urea nitrogen synthesis rate was measured in relation to intravenous alanine infusion (constant
infusion rate of 2 mmol/kg/h for 4.5 hours13 after a 12-hour
fast. Blood samples were obtained from a vein of the contralateral arm every 45 minutes, starting 90 minutes before
alanine infusion. A final blood sample was obtained 90 minutes after alanine infusion was discontinued. Urine was collected quantitatively by voiding in five consecutive 90-minute
periods (every second blood sampling). Subjects were not fed
in the course of the test.
During the experiment, urine flow was stimulated by peroral water or saline infusion to keep diuresis above 2 mL/
min. This was attained in nearly all subjects (mean diuresis,
2.8 mL/min), and diuresis was neither different in paired
experiments (2.5 and 3.1 mL/min at entry into the study and
after 3 months, respectively) nor in the two groups. The total
amount of water and saline administered in paired experiments was approximately the same, i.e., É2,000 mL. Mild
fluid retention was observed in a few experiments but never
exceeded 1 L (õ2.5% of body water). There were no side effects or complications during the infusion of alanine. In particular, no subject complained of nausea or vomited.
The urea-N synthesis rate (UNSR) during each 90-minute
period was measured as the sum of urea-N excretion rate in
urine and accumulation of urea-N in the urea space, assumed
to equal total body water (TBW), as14
UNSR Å (E / A)/(1 0 L)
where E Å (Urine flow, L/h) 1 (Urinary urea-N, mmol/L); A
Å (Change in blood urea-N, mmol/L/h) 1 (TBW, liters); L
Å (Fractional loss of newly formed urea in the gut). TBW
was considered equal to the distribution space of antipyrine,15
calculated in both conditions in the course of the antipyrine
clearance test. Intestinal loss of urea-N due to bacterial hydrolysis was taken to be 0.26.16
In each experiment, the FHNC was calculated as the slope
of the linear regression of UNSR on the corresponding average a-amino-N concentration during each time period (mean
of a-amino-N values measured at the beginning and at the
end of each urine collection) (Fig. 1).
In all cases, the galactose elimination capacity (GEC) was
measured according to Tygstrup’s technique17 and antipyrine
clearance by means of a two-sample procedure.18
In our laboratory, the normal values of galactose elimination capacity are greater than 6.0 mg/kg/min,19 antipyrine
clearance is greater than 30 mL/min,18 and the functional
hepatic nitrogen clearance is greater than 25 L/h.20 Repeated
measurements of the three tests in the same subject vary
within {10%, {8%, and {15%, respectively.20
Encephalopathy was quantitatively measured by means
of psychometric tests. NCT was performed according to the
method of Conn,11 whereas the evaluation of CRTs12 was
based on the mean number of reaction times exceeding 400
milliseconds in two repeated series of 100.21 In normal subjects the time to perform the NCT is less than 50 seconds
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FIG. 1. The dynamics of a-amino-N to urea-N conversion in the
experimental group in relation to zinc supplementation are shown
(before zinc, open circles; after zinc, closed circles). Urea-nitrogen
synthesis rate increases with increasing a-amino-N concentrations
and the slope of the regression is the functional hepatic nitrogen
clearance. The continuous lines represent the average regression in
a range of a-amino-N concentrations attained in the course of the
experiments. The equations of the regressions are as follows: before
zinc supplementation, UNSR Å 026.6 / 20.9 1 a-amino-N; after
zinc supplementation, UNSR Å 027.5 / 25.3 1 a-amino-N.
and the number of CRTs exceeding 400 milliseconds is less
than 15.21
Because of the antioxidant properties of zinc,1 the amount
of lipid hydroperoxides present in plasma at the beginning
and at the end of alanine infusion was also measured as the
total concentration of thiobarbituric acid reacting substances
(TBARS).22
Laboratory Procedures. Urea-N in plasma and urine was
measured by the urease Berthelot method.23 Alanine was
measured enzymatically,24 and total a-amino-N was measured by by the dinitrofluorobenzene method.25 All analyses
were performed in batches, in duplicate or triplicate to minimize the analytical error. The intra-assay coefficients of variation are as follows: urea, {1.5%; a-amino-N, {2%; and alanine, {3%. Plasma amino acid profile was measured by
ninhydrin reaction after ion-exchange chromatography at
baseline and at the end of alanine infusion,26 with a coefficient of variation less than 5%. Plasma glucagon and insulin
levels were measured by radioimmunoassay (Glucagon and
Insulin kits; Biodata-Serono, Guidonia, Italy). Glucose levels
were measured enzymatically. Plasma zinc levels were measured by mass spectrometry.
Galactose levels were determined enzymatically (Test
Combination Galactose; Boehringer GmbH, Mannheim, Germany). Antipyrine levels were measured by an high-performance liquid chromatography technique.27
TBARS were measured using the high-performance liquid
chromatography method of Wong et al.22 with minor modifications. After separation on a C18 column, the malondialdehydeTBAR adduct was quantified using spectrofluorometry, with
excitation wavelength of 518 nm and emission wavelength of
547 nm.
Statistical Analysis. Linear correlation analysis between
variables was performed by the least squares’ method. Differences between data were analyzed by paired and unpaired t
test, whenever appropriate. Differences in serial determination of the same parameters in paired experiments were also
tested for significance using repeated-measures analysis of
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TABLE 2. Glucose, a-Amino-N, Insulin, and Glucagon
Concentrations at the Beginning (time 0) and at the End of
Alanine Infusion (time 270) in the Course of the Paired
Experiments Performed in Cirrhotic Patients Before and
After Zinc Sulfate Supplementation
Experimental Group
Time 0
Time 270
Basal experiment
Glucose (mmol/L) 6.0 (1.8) 5.3
a-Amino-N (mmol/
L)
2.2 (0.3) 8.3
Insulin (pmol/L)
59 (18)
84
Glucagon (pmol/L) 107 (78) 199
Control Group
Time 0
Time 270
(1.2)
5.2 (0.5)† 5.4 (0.7)
(1.1)*
(26)*
(99)*
2.4 (0.6)
65 (17)
104 (37)
8.4 (1.4)*
104 (32)*
183 (61)*
After 3 months
Glucose (mmol/L) 5.6 (0.8) 5.2 (0.7)
5.2 (0.5)† 5.5 (0.7)
a-Amino-N (mmol/
L)
2.1 (0.4) 7.6 (1.0)*‡ 2.5 (0.9)† 8.9 (2.0)*†
Insulin (mmol/L)
84 (26)‡ 110 (40)*
67 (14)† 104 (36)*
Glucagon (mmol/
L)
58 (32)‡ 118 (47)*‡ 101 (39)† 195 (47)*†
NOTE. Values shown are mean (SD). Normal values: fasting insulin, õ60; fasting glucagon, õ45.
* Significantly different from time 0 value.
† Significantly different from the corresponding value in the active
treatment group.
‡ Significantly different from the corresponding value in the basal
experiment.
variance. All analyses were performed on a personal computer by means of StatView II program (Abacus Concepts,
Inc., Berkeley, CA). Data in text, tables, and figures are
shown as mean (SD).
RESULTS
Plasma zinc concentration was low normal or reduced in all patients and in both groups (range, 53 to 84
mg/dL; Table 1). Oral zinc supplementation increased
plasma zinc by 60% to 109 (SD, 25) mg/dL in the experimental group (P õ .001), whereas in the control group
plasma zinc was unchanged at the end of the observation period (69 [16] mg/dL).
In the basal experiment, fasting a-amino-N, glucose,
and insulin concentrations were in the normal range,
without differences between groups. Glucagon was an
approximately twofold increased (Table 2). Alanine infusion increased a-amino-N levels fourfold and glucose
did not change significantly, whereas insulin increased
by 30% to 40% and glucagon doubled. Zinc supplementation nearly halved basal glucagon and the glucagon
response to alanine infusion in the experimental group,
whereas basal insulin increased by nearly 30%. In the
control group, fasting and alanine-stimulated insulin
and glucagon concentrations at the end of the study
period were similar to those observed in the basal experiment.
Basal ammonia levels were 30% increased in comparison with normal values in both groups (Table 3), and
doubled after alanine infusion. In the experimental
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group, zinc treatment reduced basal ammonia by 25%
and the ammonia response to alanine by 30%.
TBW, estimated by antipyrine distribution space,
was on average 42.8 (4.6) L in our patients (corresponding to 61% of body weight) and not different between
groups. It did not change at the end of the study period
(43.6 [6.9] L).
Basal UNSR was similar in paired experiments. In
the course of alanine infusion, UNSR increased linearly
with increasing a-amino-N concentrations in each experiment, the R2 coefficient of determination of linear
regression was in the range 0.77 to 0.99. In the experimental group, after zinc supplementation, amino acid–
stimulated UNSR was 15% to 20% higher, in spite of
10% lower plasma a-amino-N concentrations (Table 4).
Urinary urea excretion accounted for approximately
60% to 65% of total urea formation; the percentage was
not different in paired experiments.
FHNC was decreased in both groups of patients with
cirrhosis, in comparison with normal values of our laboratory, and increased by 25% after zinc supplements
in the experimental group. The effects of zinc on FHNC
were variable (range, 2.2 to 7.7 L/h) but observed in all
patients (Fig. 2). There were no differences between
cirrhosis of alcoholic origin (4.0 [2.5] L/h) and cirrhosis
of viral origin (4.7 [1.3]). No changes in FHNC were
observed in the control group.
In the experimental group, NCT improved by 16%
after zinc supplementation but remained abnormal in
5 of 8 cases, whereas the number of reaction times to
sound greater than 400 milliseconds decreased by 52%,
and at the end of the observation period it was abnormal only in two cases (Fig. 3). In the control group both
psychometric tests did not change significantly.
The Child-Pugh score improved significantly after
oral zinc, from values ranging from 6 to 13 to values
between 5 and 11. Among routine liver function tests,
only prothrombin activity improved significantly, but
there was a trend toward increased albumin and decreased bilirubin levels. Alkaline phosphatase activity
increased from 257 (SD, 91) U/L to 300 (81); P õ .05.
GEC and antipyrine clearance improved slightly in the
experimental group, from 1.33 (0.21) mmol/min to 1.49
(0.30) (by 12%) and from 20.5 (4.3) mL/min to 22.3 (4.2)
(by 9%), respectively. In the control group, both routine
and dynamic tests of liver function were on average
unchanged at the end of the observation period, but
there was a trend toward progressive deterioration.
The Child-Pugh score ranged between 6 and 11 at the
beginning of the observation period and between 7 and
12 after 3 months.
Plasma amino acids, both basal and alanine-stimulated, were not different in the paired experiments (not
reported in details), with notable exceptions in urea
cycle amino acids in zinc supplemented patients (Table
3). Such changes were not observed in the control
group.
In the experimental group, fasting TBARS were 1.12
(SD, 0.56) mmol/L in the basal experiment, i.e., approximately twice that of control values, and increased by
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TABLE 3. Plasma Concentrations of Urea, Ammonia, and Amino Acids Involved in Urea Formation at the Beginning (time
0) and at the End of Alanine Infusion (time 270) in the Course of the Paired Experiments Performed
in Cirrhotic Patients Before and After Zinc-Sulphate Supplements
Experimental Group
Time 0
Basal experiment
Urea (mmol/L)
Ammonia (mmol/L)
Glutamine (mmol/L)
Ornithine (mmol/L)
Citrulline (mmol/L)
Arginine (mmol/L)
After 3 months
Urea (mmol/L)
Ammonia (mmol/L)
Glutamine (mmol/L)
Ornithine (mmol/L)
Citrulline (mmol/L)
Arginine (mmol/L)
Control Group
Time 270
Time 0
Time 270
5.1
42
407
122
23
102
(1.1)
(17)
(79)
(35)
(10)
(24)
7.3
96
693
114
53
97
(1.0)*
(23)*
(146)*
(28)
(22)*
(28)
5.7
45
369
102
31
93
(2.0)
(24)
(117)
(27)
(12)
(20)
7.5
92
603
90
71
98
(2.2)*
(20)*
(162)*
(13)
(15)*
(26)
4.9
34
323
91
21
73
(0.9)
(16)
(77)
(41)
(6)
(19)‡
7.0
65
582
81
105
122
(1.3)*
(26)*‡
(150)*
(17)‡
(21)*‡
(22)*‡
5.3
54
383
93
39
88
(1.7)
(22)†
(57)
(24)
(18)
(33)
7.2
101
558
88
71
90
(1.7)*
(31)*†
(175)
(33)
(32)*
(21)
NOTE. Values shown are mean (SD).
* Significantly different from time 0 value.
† Significantly different from the corresponding value in the active treatment group.
‡ Significantly different from the corresponding value in the basal experiment.
29% (1.44 [0.68]; P õ .05) during alanine infusion. After
zinc supplementation, fasting TBARS were not
changed, but did not increase further in response to
alanine (Fig. 4). In the control group alanine infusion
was followed by a marked increase of TBARS in both
experiments at the beginning and at the end of the
observation period.
In the two patients of the experimental group, in
which all tests were repeated approximately 6 months
after the end of zinc supplementation, plasma zinc levels returned to pretreatment values after zinc withdrawal (Table 5). This was accompanied by a decrease
in FHNC to values similar to those observed before
treatment, an increase in fasting ammonia levels and
in the ammonia response to alanine, a decrease in fasting and stimulated insulin, and an increase in glucagon. Clinically, there was a deterioration in psychometric tests, whereas routine and dynamic laboratory data
returned toward pre–zinc treatment levels.
DISCUSSION
Our study indicates that long-term oral zinc supplementation increases the hepatic conversion of amino
acids into urea. This was associated with an objective
clinical and biochemical improvement, not limited to
the performance of psychometric tests or to liver func-
TABLE 4. Average a-Amino-N Concentrations and UNSR in Each Time Period
in the Course of the Paired Experiments, and Functional Hepatic Nitrogen Clearance
Experimental Group
Period
(min)
090 to 0
0 to 90
90 to 180
180 to 270
270 to 360
Basal
a-AN (mmol/L)
UNSR (mmol/h)
a-AN (mmol/L)
UNSR (mmol/h)
a-AN (mmol/L)
UNSR (mmol/h)
a-AN (mmol/L)
UNSR (mmol/h)
a-AN (mmol/L)
UNSR (mmol/h)
2.4
16
4.2
75
6.8
120
7.9
136
5.8
82
(0.3)
(9)
(0.3)
(18)
(0.9)
(17)†
(0.9)
(32)
(1.0)
(21)
FHNC (L/h)
20.9 (3.9)
After 3 Months
2.2
18
3.9
85
6.3
142
7.3
154
5.8
105
(0.3)†
(14)
(0.5)
(19)
(1.0)†
(36)†
(1.2)†
(27)†
(1.1)†
(19)*†
25.3 (3.8)*†
Control Group
Basal
2.5
21
4.4
68
7.0
106
7.8
132
5.8
83
(0.6)
(14)
(0.6)
(16)
(0.9)
(31)
(1.1)
(18)
(1.0)
(16)
2.6 (1.0)
22 (13)
4.3 (1.0)
76 (20)
7.0 (1.5)
113 (17)
8.3 (1.8)
132 (22)
6.6 (1.7)
96 (6)
20.0 (2.9)
17.9 (3.4)
NOTE. Values shown are mean (SD).
a-amino-N concentrations are the average of values obtained at the beginning and at the end of each time period.
* Significantly different from the corresponding value in the basal experiment.
† Significantly different from the corresponding value in the control group.
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MARCHESINI ET AL.
FIG. 2. Functional hepatic nitrogen clearance in the experimental group before and after zinc supplementation (A) and in the control
group (B). The values measured in individual subjects in paired experiments are connected by a continuous line. Average values are
indicated by open circles and dotted line.
tion but also expressed by the comprehensive score of
Child-Pugh.
In keeping with a previous observation,5 we found
that plasma zinc concentrations of cirrhotic patients
return to normal after treatment with 600 mg zinc sulphate for 3 months. The recommended dietary allowances of zinc are 15 mg in males and 12 in females,28
25% being absorbed,29 and urinary zinc losses are negligible in controls and as high as 4 mg/d in patients with
liver disease.30 The doses and long-term treatment we
FIG. 3. (A) Number connection test (NCT) and (B) number of
continuous reaction times to sound ú400 msec (CRT-s) in cirrhotic
patients at the beginning (h) and at the end of the study period (j).
*Significantly different from the corresponding pretreatment value.
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1089
FIG. 4. Plasma concentration of lipid peroxides in the fasting
state (time 0*) (h) and at the end of alanine infusion (time 270*) (j)
in the two groups of cirrhotic patients at the beginning (Base) and
at the end (3-month) of the study period. *Significantly different
from the corresponding time 270* value before zinc supplementation
in the experimental group and time 270* value in both experiments
in the control group.
used are likely to influence positively hepatic zinc content, which is known to be reduced by approximately
50% in patients with liver disease despite low tissue
zinc turnover.31 Accordingly, the activity of the serum
zinc-dependent enzyme alkaline phosphatase increased during oral supplementation, as previously reported.4 Unfortunately, as shown in two patients of the
experimental group, zinc levels rapidly decreased after
treatment withdrawal, which makes continuous supplementation mandatory.
The return to normal of plasma zinc levels in the
experimental group was associated with a remarkable
increase in the hepatic conversion of amino acids into
urea and decreased concentrations of amino acid–stimulated ammonia, which was not observed in the control
group, carefully matched for liver cell failure and hepatic encephalopathy. The data expands previous evidence first reported by Reding et al. in cirrhotic patients with chronic hepatic encephalopathy,5 in which
only the basal concentration of urea and ammonia was
measured.
The methodology of the present study, i.e., the measurement of the dynamics of hepatic urea formation
during standardized conditions of substrate availability,9 is the same previously used to measure the effects
of hormones or drugs in several conditions. The assumptions underlying the technique have been extensively dealt with in previous papers.9,20 In the calculation of UNSR, intestinal hydrolysis was considered a
fixed fraction of total urea nitrogen excretion on the
basis of the average values derived from the literature.16 In our study, all patients were taking lactulose
at the time of study, which reduces gut urea hydrolysis.32 This may cause overestimation of urea synthesis
rate, but it is not likely to be of relevance in paired
experiments, because no changes in lactulose treatment occurred, and zinc is not expected to affect intestinal hydrolysis of urea per se.
Zinc supplementation resulted in a significant increase in FHNC, which graphically corresponds to a
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1090 MARCHESINI ET AL.
HEPATOLOGY May 1996
TABLE 5. Laboratory Data in the Two Patients of the Experimental Group (cases 7 and 8) in Which
all Tests Were Performed Before Entry Into the Study, at the End of the 3-Month Zinc Supplementation
And 6 Months After the End of Treatment
Before Zn
Plasma Zn (mg/dL)
FHNC (L/h)
Ammonia (mmol/L)
Basal
End of alanine
Insulin (pmol/L)
Basal
End of alanine
Glucagon (pmol/L)
Basal
End-of-alanine
NCT (s)
CRT-s ú400 ms (%)
GEC (mg/kg/min)
Alkaline phosphatase (U/L)
After Zn
Case 7
Case 8
84
25.3
63
26.5
After 6 Months
Case 7
Case 8
105
31.0
95
30.2
Case 7
Case 8
75
25.1
72
24.7
18
52
61
111
17
26
35
91
28
73
48
144
57
86
43
93
107
122
86
138
43
72
43
86
41
93
100
8
1.83
297
53
114
35
2
1.80
307
116
200
120
26
1.32
179
60
139
60
10
1.55
229
100
160
102
15
1.41
213
74
129
48
7
1.69
240
Individual values are reported for cases 7 and 8, respectively.
counterclockwise shift of the relationship of a-aminoN to urea-N, i.e., more urea was produced at any aamino-N concentration (Fig. 1). Several factors are
known to regulate the kinetics of the process and might
theoretically be responsible for the effects of zinc on
urea synthesis.
Glucagon is the most potent stimulatory drive for
hepatic amino acid conversion and urea synthesis in
normal subjects,33 and also mediates the effects of other
hormones, namely cortisol and cathecolamines.9 It
stimulates urea synthesis by increasing amino acid
transport in the liver and through up-regulation of urea
cycle enzymes,34,35 but its effects are blunted or absent
in cirrhosis.14,36 In the present study, zinc supplementation was associated with an unexpected, systematic
inhibitory effect on basal and alanine-stimulated glucagon concentration, excluding any glucagon-mediated
effect on hepatic nitrogen clearance. Also insulin levels
changed after zinc, with basal insulin increasing by
30%, but hyperinsulinemia has a modest down-regulatory effect on urea synthesis.37
A second determinant of hepatic nitrogen clearance
is liver cell function, in both acute38 and chronic liver
disease.13 In experimental animals, zinc stimulates a
variety of metabolic reactions that protect the liver
from the hepatotoxic activity of drugs and toxins.3 In
humans, zinc deficiency is associated with decreased
plasma levels of proteins synthetized by the liver,
which are corrected by zinc supplementation.39 In the
present series, long-term oral zinc administration was
accompanied by a remarkable improvement of both
routine and dynamic liver function tests of an order of
10%, whereas FHNC improved by 25% and reached
values usually measured in normal subjects (maximum
value, 31 L/h), in spite of the advanced condition of
cirrhosis with actual or previous encephalopathy. Also
the Child-Pugh score decreased, and all patients re-
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ported a subjective improvement in their general conditions. Zinc deficiency induces anorexia40 and taste
abnormalities,41 which are reversed by zinc supplementation.41 Dietary intake in our patients was not controled so strictly to exclude that at least part of the
general improvement might be caused by changed nutritional habits. However, in this case hyperglucagonemia would be expected, because an increase in
dietary proteins augments a-amino-N to urea conversion42 through glucagon stimulation, which in turn activates urea cycle enzymes.34,35
Theoretically, the improvement in FHNC might also
be the effect of spontaneously fluctuating disease activity and/or alcohol abstinence in alcoholic cirrhosis, in
which liver function considerably improves in the first
1 to 2 years after alcohol abstinence.43 However, no
specific effect of alcohol was proven, because FHNC
increased in all subjects in the experimental group,
irrespective of the origin of the disease (alcohol or hepatitis C virus), whereas in the control group no significant changes were observed. A specific effect of zinc
treatment is further supported by a longer follow-up
in two patients of the experimental group, in whom
zinc withdrawal and the return of plasma zinc to pretreatment levels was accompanied by a reduction in
FHNC and metabolic changes opposite to those observed during zinc treatment.
Another possible mechanism for the effects of zinc
on hepatic urea production might be its potential activity as an antioxidant, which has been postulated in
a few chemical systems.1 Scavenger systems, such as
glutathione,44 are reduced in cirrhosis, mainly in disease of alcoholic origin. Pharmacological doses of zinc
in vivo have been shown to protect against hepatic toxicity in experimental animals,45 and there is evidence
that zinc deficiency may increase the susceptibility of
the liver to oxidative damage.1 Although fasting
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HEPATOLOGY Vol. 23, No. 5, 1996
MARCHESINI ET AL.
TBARS concentrations were increased both before and
after zinc supplements, as well as in the control group,
the increase of TBARS in response to the metabolic
stress caused by alanine infusion was abolished after
zinc supplementation. Data are needed to clarify the
mechanism(s) by which zinc may act as an antioxidant,
and its potential interference with hepatic metabolism.
The most likely explanation for increased FHNC in
response to zinc supplements remains a direct action
of zinc at subcellular level on urea cycle enzyme. In
vitro studies have shown that zinc plays a regulatory
action on OTC (EC 2.1.3.3),46,47 a key enzyme for urea
synthesis in the liver. In vivo, experimental zinc deficiency in rats decreases the activity of OTC in rats,48
and zinc supplementation in zinc-deficient cirrhotic
rats produces a remarkable increase in hepatic OTC,
which parallels the increase in serum and hepatic zinc.8
We observed that plasma levels of individual urea cycle
amino acids beyond the OTC step were increased,
whereas ornithine concentrations (before the OTC
step) were reduced after zinc supplementation in the
control group only when compared with pretreatment
values, in keeping with a putative action of zinc on
enzyme activity.
In rats48 and humans,4,49 zinc deficiency is accompanied by increased serum ammonia levels, and zinc supplementation reduces ammonia in experimental animals and in man.4,5,8 Zinc deficiency was also reported
to affect the activity of muscle glutamine synthetase,50
which also leads to hyperammonemia. In the present
study, fasting plasma ammonia was not significantly
reduced by zinc treatment, but the ammonia increase
in response to alanine, which always occurs during infusion, was reduced on average from 54 (SD, 19) to only
30 mmol/L.16 Fasting ammonia is the result of several
factors, including the antecedent protein intake, bowel
movements, and lactulose therapy, whereas the ammonia response to alanine strictly depends on the ability
of the hepatic parenchyma to dispose of the ammonia
generated in amino acid metabolism.
It might be argued that reduced ammonia might also
derive from enhanced renal ammonia excretion, because the kidney is involved in ammonia disposal via
glutamine uptake.51 Hyperammonemia shifts ammonia disposal by the kidney from venous ammonia release to urinary ammonia excretion. Urinary ammonia
was not measured, but no differences in glutamine concentrations were observed, and ammonia levels were
lower after zinc.
It is not possible to speculate which is the origin of
the improved mental state after zinc treatment, reported here as well as in several previous studies5,7
but not in others.6,52 They do not seem to derive from
changes in fasting amino acid profile or in the ratio of
branched-chain to aromatic amino acids, which
changed little from 1.86 (SD, 0.64) to 2.06 (0.73). In
particular, it is not known whether they are mediated
by changes in ammonia, or stem from a direct action
of zinc on the central nervous system. The improvement in psychometric testing was only observed in the
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experimental, zinc-supplemented group and not in the
historical control group, which seems to exclude a
chance result due to disease variability in chronic hepatic encephalopathy. It was also independent of the
lowering effects on ammonia. Improved mental state
might simply result from the general positive trend in
hepatic metabolic activities, nutrition, and well being
observed in patients in the experimental group. Such
positive effects of zinc supplementation deserve further
analysis in larger, randomized, double-blind studies, in
which the metabolic effects of zinc on urea synthesis
might also receive definite validation. However, the advantages of zinc supplementation must be balanced
against the potential hazard of zinc toxicity, namely
anemia and neutropenia, caused by zinc-induced enterocyte metallothionein synthesis and copper deficiency,3 which were not observed in our patients.
Acknowledgment: We are indebted to Dr. Silvia Maselli and Dr. Tiziano Mussi, Farmacia, Policlinico S.
Orsola-Malpighi, Bologna, for kindly preparing the alanine solution and the zinc tablets used in the present
experiments; to Dr. Rita Flamia, Laboratorio Centralizzato, Policlinico S. Orsola-Malpighi, Bologna, for
hormone determination; to Dr. Anna Zapparoli, Presidio Multizonale di Igiene e Profilassi, Azienda Ospedaliera Città di Bologna, for the determination of plasma
zinc levels; and to technician Raffaela Chianese for assistance.
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