Download Therapeutic efficacy of L‐ornithine‐L‐aspartate infusions in patients

Therapeutic Efficacy of L-Ornithine-L-Aspartate Infusions in
Patients With Cirrhosis and Hepatic Encephalopathy:
Results of a Placebo-Controlled, Double-Blind Study
GERALD KIRCHEIS,1,2 RÜDIGER NILIUS,1,3 CONSTANTIN HELD,4 HANS BERNDT,4 MICHAEL BUCHNER,5 ROMAN GÖRTELMEYER,2
RÜDIGER HENDRICKS,6 BERTRAM KRÜGER,7 BODO KUKLINSKI,5 HELMUT MEISTER,8 HANS-JÜRGEN OTTO,8
CHRISTOPH RINK,1,9 WOLFGANG RÖSCH,10 AND SABINE STAUCH10
One hundred twenty-six patients with cirrhosis, hyperammonemia (ú50 mmol/L), and chronic (persistent)
hepatic encephalopathy (HE), which developed spontaneously without the existence of known precipitating
factors, were enrolled in a randomized, double-blind,
placebo-controlled clinical trial of intravenously administered L-ornithine-L-aspartate (OA). Patients with subclinical (grade 0, West-Haven criteria) hepatic encephalopathy (SHE), characterized by a prolonged number
connection test A (NCT-A) time, and manifest HE (grades
I and II, West-Haven criteria) were included in the investigation. The trial was planned as a confirmatory clinical
trial. OA administered in a dose of 20 g/d, as well as
placebo, were dissolved in 250 mL of 5% fructose and
infused intravenously for a period of 4 hours during 7
consecutive days with a superimposed protein load at
the end of the daily treatment period. Primary variables
were postprandial venous ammonia and NCT-A performance time measured following OA or placebo infusions
to evaluate the net effect of the treatment on the prevention of the protein-induced hyperammonemia, and on
parameters such as NCT-A influenced by hyperammonemia. Mental state gradation, portal systemic encephalopathy index (PSEI), and fasting ammonia levels were
estimated as additional efficacy parameters. The data
presented are based on the total study sample (intent-totreat analysis), which included 63 patients in the placebo
group and 63 patients in the OA group. Of the 126 patients, 114 met all the criteria for inclusion and completed the trial and treatment as outlined in the protocol
(treated-per-protocol analysis). During baseline, the placebo and treatment groups were homogeneous with regard to mental states, NCT-A performance time, fasting
venous blood ammonia levels, and Child-Pugh criteria.
Although a slight improvement occurred in the placebo
group, NCT-A performance times (P õ .001) and post-
Abbreviations: HE, hepatic encephalopathy; SHE, subclinical hepatic encephalopathy;
OA, L-ornithine-L-aspartate; GS, glutamine synthetase; NCT-A, number connection test A;
PSEI, portal systemic encephalopathy index.
From the 1Martin-Luther-University Halle-Wittenberg, Department of Internal Medicine, Halle; 2Merz Clinical Research Department and Merz Biostatistics Department,
Frankfurt/Main; 3Hospital ‘‘Max Uibeleisen,’’ Department of Internal Medicine, Bad Kissingen; 4Humboldt University, Department of Internal Medicine ‘‘Theodor Brugsch,’’ Charité, Berlin; 5Hospital, Department of Internal Medicine, Rostock; 6Hospital, Department of
Internal Medicine, Heide; 7Hospital ‘‘Fränkische Schweiz,’’ Department of Internal Medicine, Ebermannstadt; 8Hospital ‘‘St. Vincenz,’’ Department of Internal Medicine, Heiligenstadt; 9Hospital, Department of Internal Medicine, Aue; and 10Hospital ‘‘Nordwest,’’ Department of Internal Medicine, Frankfurt/Main, Germany.
Received January 4, 1995; accepted February 25, 1997.
Address reprint requests to: Gerald Kircheis M.D., Merz Clinical Research Department,
Eckenheimer Landstrasse 100-104, 60318 Frankfurt/Main, Germany. Fax: /49 691503409.
Copyright q 1997 by the American Association for the Study of Liver Diseases.
0270-9139/97/2506-0010$3.00/0
prandial venous ammonia concentrations in the OAtreated group showed improvements in comparison
with placebo. In addition, venous fasting blood ammonia
concentration (P õ .01), mental state gradation (P õ
.001), and PSEI (P õ .01), which includes the mental state
gradation, NCT-A time, and postprandial venous ammonia in this trial, improved to a much higher degree in
the OA group than in the placebo group. In subgroups
retrospectively classified according to their initial mental state gradation, OA showed differential but uniformly significant efficacies in patients with manifest
HE with respect to ammonia-lowering, improvement in
NCT times, and mental state gradation. In patients with
initial SHE, OA revealed differences between the medications in the psychometric test used. Adverse events
consisting of mild gastrointestinal disturbances were
observed in 3 of the OA-treated patients (5%). OA infusion appears to be a safe, effective treatment of chronic
(persistent) manifest HE in cirrhotic patients. Additional investigations are required to assess the efficacy
of OA in patients with SHE, as well as in patients with
more severe grades of HE. (HEPATOLOGY 1997;25:13511360.)
Hepatic encephalopathy (HE) is one of the major complications of cirrhosis. Five years after the diagnosis of cirrhosis,
the probability of developing at least one episode of this specific form of decompensated cirrhosis is in the range of 26%.1
Once clinical decompensation has occurred, however, the
prognosis (16%1 to 22%2 probability of survival at 5 years)
compared with a survival probability of 55%2 to 70%1 in cirrhotic patients without HE is very poor.1,2 Therefore, prevention and effective treatment of HE may have important prognostic implications in cirrhotic patients.
Hyperammonemia3,4 by a variety of postulated mechanisms3-8 is felt to be one of the primary pathogenetic factors
in the development of HE.8 The majority of therapeutic measures currently in use are therefore directed at reducing blood
ammonia levels,9-13 mainly by diminishing enteric ammonia
production. Otherwise, it is known that liver and muscle play
an important part within the ammonia detoxification system,
especially in cirrhosis. Irrespective thereof, increased extraintestinal ammonia production and reduced ammonia detoxification capacity seen in patients with cirrhosis14-16 and
chronic (persistent) manifest HE, as well as subclinical hepatic encephalopathy (SHE), have, to date, not been targets
of therapeutic agents within randomized, placebo-controlled
trials.
Results of animal experiments13-23 suggest that L-ornithine-L-aspartate (OA) may have a favorable influence on HE
by virtue of its blood ammonia–lowering effects. A series of
investigations in human subjects that started more than 25
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years ago has confirmed these suggestions and has shown
that OA can reduce blood ammonia and improve the symptoms and laboratory features of HE.24-27
Experimental and clinical findings in liver, muscle, and
brain have provided new insights into the mechanisms of
action of these ‘‘ammonia-lowering’’ amino acids, especially
in the therapy of HE. These tissues are important target
organs in the removal of ammonia in hyperammonemia.1416,20-22,28-32
It has been proposed that, whereas aspartate and
citric dicarboxylates under pathological condition in cirrhotic
patients may serve as a carbon source for the impaired glutamine synthetase (GS) flux in the perivenous scavenger hepatocytes,15,31-36 ornithine improves the flux through the impaired urea cycle enzyme system, especially through
carbamylphosphate synthetase, localized in periportal hepatocytes.20-23 In contrast to hepatic ammonia removal by urea
and glutamine synthesis,14-16,20-23,31,32 recent findings suggest
fundamentally different regulatory mechanisms for GS27,37-39
and removal of excess ammonia in muscle27,37 and brain38,39 —
tissues that are devoid of an effective urea cycle.30
These concepts are the basis for the use of amino acids
such as ornithine to stimulate the impaired flux of the urea
cycle20-23 and to interact with brain glutamine,38,39 as well as
dicarboxylates and amino acids such as a-oxoglutarate or
aspartate, benzoate, and malate to stimulate the impaired
flux of glutamine synthesis in liver34-36 and muscle27,37 of cirrhotic patients.
To assess the clinical efficacy of OA in the treatment of HE,
a randomized, double-blind, placebo-controlled, multicenter
trial with intravenously administered OA in cirrhotic patients with hyperammonemia and chronic (persistent), manifest HE or SHE was performed. The aim was to evaluate in
a practice-adapted design the influence of OA on the improvement of number connection test A (NCT-A) times and of mental states as a consequence of the ammonia-lowering effect.
PATIENTS AND METHODS
Inclusion criteria were assessed 16 to 24 hours before the start of
the first infusion. Patients who met the following inclusion criteria
assessed at the randomization (day 01) were eligible for the study:
1) chronic (persistent), manifest HE (mental state grade I or II, according to the West-Haven criteria9,11,40 developed spontaneously
without existence of specified precipitating factors or SHE (mental
state grade 0 and an NCT-A performance time ú 30 seconds);9,11,40
2) hyperammonemia (venous ammonia concentration ú 50 mmol/
L);9,11 and 3) cooperative, hospitalized, adult patients with histologically proven cirrhosis or overt, clinically proven cirrhosis confirmed
ultrasonographically who volunteered to remain in the hospital for
the full 7 days of the investigation.
Patients were included if they remained stable with respect to HE
grade, existence of hyperammonemia, and increased NCT-A times
between randomization (day 01) and first treatment day (day 0).
Exclusion criteria were the following: 1) active, major complications of portal hypertension, such as gastrointestinal hemorrhage,
hepatorenal syndrome, or spontaneous bacterial peritonitis; 2) acute
superimposed liver injury; 3) serious nonhepatic diseases such as
decompensated heart failure and/or respiratory failure, decompensated diabetes mellitus, renal insufficiency, or electrolyte imbalances; 4) Acute (recurrent), severe HE (mental state grade 3 or
4);9,11,40 5) pregnancy or lactation; 6) age ° 18 years; 7) contraindications to intravenous infusion of fructose such as fructose intolerance,
hyperhydration, hypotonic dehydration, hypokalemic alkalosis, or
acidosis; 8) administration of anti-HE medications such as neomycin,
lactulose, lactitol, branched-chain amino acids, etc.; and 9) any additional precipitating factors such as high protein intake (additional
high-protein meals), constipation or intake of psychostimulants, sedatives, antidepressants, benzodiazepines, or benzodiazepine-antagonists.
Study Medications
Study medications consisted of intravenous infusions of 20 g OA
(4 ampules of 10 mL each) in 5% fructose (250 mL) administered
daily over 4 hours (8:00 AM until 12:00 PM) or the placebo solution
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(4 ampules of 10 mL each in 250 mL 5% fructose) for 7 consecutive
days. The placebo solution contained riboflavin and polyethylene glycol to make the control solution identical to the OA infusate in appearance, behavior, and viscosity. All patients were given a nutritious diet containing 1 g meat-vegetable dietary protein per kg body
weight per day. The quantity of protein was divided at the test days
among three main meals of the day in such a way that, as in a
previous study,27 the quantity of protein consumed with breakfast
was 0.25 g/kg body weight and that consumed with lunch was 0.5 g/
kg body weight. In this way, ingestion of an identical amount of
protein before determination of the postprandial ammonia, the NCTA times, and the mental state at 1:00 PM was guaranteed.
Concomitant medications that the patients were receiving and
that were continued throughout the study included diuretic drugs
(OA: 24 patients; placebo: 18 patients) and/or aldosterone antagonists (OA: 18 patients; placebo: 16 patients), b-adrenergic blocking
agents (OA: 6 patients; placebo: 4 patients), and digitalis-containing
drugs (OA: 4 patients; placebo: 5 patients). Long-term administration
of either clomethiazole (4 patients), a drug used for treatment of
alcohol-withdrawal syndrome, or a tranquilizer (1 patient) was also
continued.
Therapeutic Endpoints
The size of comprehensive test batteries renders precise, defined
therapeutic endpoints impractical for use in the clinical situation
and for follow-up observation in large therapeutic studies. Consequently, the participants of the study group decided to use a limited
number of simple, validated, sensitive test procedures.
Primary variables were postprandial venous ammonia and NCTA performance time representing the net effect of the previous 4hour infusion in combination with the effects of the previous 2 to 7
days of infusion on the prevention of the protein-induced (12:00 PM
meal [0.5 g protein per kilogram of body weight]) hyperammonemia
and on parameters such as NCT-A time, influenced by hyperammonemia. Mental state gradation and portal systemic encephalopathy
index (PSEI), assessed at the same time as the primary variables,
as well as fasting ammonia levels, representing the impact of the
previous 2 to 7 days of infusion, were estimated as additional efficacy
parameters. Any difference between the OA-and the placebo-treated
group with respect to the above-indicated parameters (NCT-A time,
NH4/, mental state gradation, PSEI) could be interpreted in terms
of a prevention of protein-induced hyperammonemia.
Inclusion criteria were checked at a nonfixed time point at the
time of randomization (day 01) before any treatment or superimposed protein load. Therefore, these parameters could not be used
as baseline parameters for the analysis of primary and secondary
response variables. The baselines of postprandial NH4/, NCT-A, and
HE-grading used in efficacy analyses were obtained after the first
OA- or placebo-infusion (day 0; 1:00 PM). From this point of view,
the therapeutic effects after 2, 4, and 7 days of treatment will be
analyzed for these parameters at postprandial time points (1:00 PM).
Because the fasting ammonia level (day 0; 8:00 AM) is the only uninfluenced baseline level, the therapeutic effects of OA- or placeboinfusion on this parameter will be evaluated after 2, 4, and 7 days
in the fasting state (8:00 AM).
NCT-A Performance Time in Seconds. We used the NCT-A, a standardized psychometric test, for the diagnosis of SHE.9,11,40 The availability of four validated parallel forms of NCT-A and the inclusion
of a trial run of a simpler form of NCT-A before the first use of the
test at the randomization point was the most reliable way of ensuring
that learning effects were minimized.
Venous Ammonia Concentration. The ammonia determination was
performed according to the enzymatic determination of ammonia
with glutamate dehydrogenase in a rapid and interference-free photometric determination (340 [334] nm) of NH4/ in ‘‘native’’ blood
plasma according to Da Fonseca-Wollheim.41 For safety reasons,
blood after withdrawal was immediately taken by refrigerated transport to the laboratory for immediate (within 15 minutes of withdrawal of the blood) determination of NH4/. Because the same standardized technique was used at all the centers, and because all the
laboratories were subject to continual quality control with method
control and checks in accordance with ‘‘Good Laboratory Practice,’’
NH4/ was determined under qualitatively consistent conditions at
all centers. Because of ethical considerations, we were not able to
use arterial blood in this investigation.
Mental State Grade. The mental state was graded on a 0-to-4 scale
of severity.9,11,40 The interindividual variation of its assessment is
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consistently of the order of 5%.9 At all centers, two physicians participating in this trial were trained in the correct use of mental state
grading according to West-Haven criteria introduced by Conn and
Lieberthal9; the mental state of an individual patient was assessed
by the same observers and the grade defined by a consensus decision.
PSEI. The PSEI according to the West-Haven criteria9,11,40 requires the systematic semiquantitative estimation of five components of the HE syndrome, each graded on a 0-to-4 scale of severity.9,11,40 Each of the parameters is arbitrarily weighted separately
and in proportion to its importance. Because calculations permit the
presentation of the percentage improvement noted in an individual
patient or in groups of patients, the use of the index is acceptable
even when measurements of specific symptoms are missing. In the
presented clinical trial, the PSEI contains venous postprandial ammonia, mental state gradation, and NCT-A time, whereas electroencephalogram monitoring and asterixis are missing. All analyzed parameters were estimated at the same time (1:00 PM).
Safety Parameters. Safety parameters included blood tests (hemoglobin, hematocrit, white blood cell count, and thrombocytes) and
liver function tests (alanine aminotransferase, aspartate aminotransferase, g-glutamyltransferase activity, serum bilirubin, serum
albumin concentrations, cholinesterase activity, prothrombin time,
and partial thromboplastin time) on days 0 and 7 (8:00 AM). The
same standard methods were used at all centers. All laboratories
were subject to constant quality control by means of suitable method
checks.
Compliance, Tolerance, and Adverse Events. The consumption of
28 ampules of OA- or placebo-infusion corresponds to 100% compliance, a minimum consumption of 26 ampules of infusion concentrate
being regarded as acceptable (95% compliance). All adverse events
were assessed by the investigator according to traditional clinicaltherapeutic methods. Any pathological clinical or laboratory findings
observed during the trial were monitored and documented until their
normalization was observed or their cause and their correlation with
the investigational medication could be explained. In the event of a
serious adverse event, the investigator was entitled to open the decoding envelope prepared for the individual patient. None of the
prepared decoding envelopes were opened.
Statistical Methods
Clinical data management was performed using a data bank established in INGRES version 6 (Relational Technology Inc., Alameda,
CA). The statistical evaluation was performed using SAS procedures
of version 6.06 (SAS Institute Inc., Cary, NC). The medication differences at the end of treatment of the target variables NCT-A and
postprandial ammonia concentration were tested by the Mann-Whitney U test (Wilcoxon two-sample test). The global level of significance
was a Å 0.05 (nominal value). The m-correction that became necessary for the assessment of the medication differences at any point
of measurement (except for day 0) was performed according to Hochberg and Benjamini.42 Further inferential analyses of additional variables (fasting ammonia concentration, mental state grade, PSEI,
etc.), as well as subgroup analyses of the indicated parameters, were
made by the Wilcoxon two-sample test, Wilcoxon signed rank test,
x2 test, Kruskal-Wallis test, Fisher’s two-tailed exact test, and a test
for homogeneity of odds ratios. The error probabilities of these tests
are descriptive significances.
Sample size calculation was based on the results of the ammonialowering effect of OA in a previous randomized, placebo-controlled,
double-blind trial.27 The calculation was based on the clinical assumption that 20 g OA/d during a 7-day infusion period was at least
equally effective in reducing blood ammonia concentration as a single
infusion of 20 or 40 g OA. The required number of patients per group
was estimated as n ú 18 (a* of 0.05/2 Å 0.025; b Å 0.10). The a was
corrected with regard to the repeated tests for two primary variables.
Randomization was performed with the random number generator
of G. Marsaglia and T. A. Bray in random permuted size blocks of
four to avoid a center- and treatment-related bias. The treatment
forms (ampules of placebo and OA) were appropriately numbered.
In accordance with the randomization list, 63 patients each were
allocated to the groups treated either with OA infusion concentrate
or a placebo. The code of the randomization list was not broken until
the last Case Report forms had reached the Biostatistical Department and the database was frozen.
Ethical Considerations
Testing a new substance against a reference substance is permissible only if the latter has been shown to induce effects above and
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TABLE 1. Baseline Data of 126 Cirrhotic Patients with
Hyperammonemia and Chronic (Persistent) Manifest Hepatic
Encephalopathy or Subclinical Hepatic Encephalopathy (SHE),
Included in the Trial (Intent-to-Treat Sample)
OA Group
No. (%)
Patients
Male
Female
Age (yr)
Body weight (kg)
Body height (cm)
Body mass index
Duration of cirrhosis
(mo)
Etiology
alcohol
posthepatitic
others
Child-Pugh grade
A
B
C
HE grade
0
SHE
I
II
Ammonia (inclusion)
NCT-A (inclusion)
Mean
Placebo Group
SD
63
45 (71)
18 (29)
No. (%)
SD
63
46 (73)
17 (27)
53.9
75.1
170.6
25.7
12.4
15.5
9.0
3.8
52.3
72.0
173.2
24.0
13.3
15.1
7.4
4.4
58.9
49.7
60.2
56.0
90.0
60.2
45.4
19.8
49 (78)
12 (19)
2 (3)
51 (81)
8 (13)
4 (6)
30 (48)
28 (44)
5 (8)
34 (54)
22 (35)
7 (11)
0
26
26
11
63
62
0
27
27
9
63
63
(41)
(41)
(17)
(100)
(98)
Mean
78.6
61.8
23.5
22.8
(43)
(43)
(14)
(100)
(100)
NOTE. The medication groups in the intent-to-treat sample were homogeneous with regard to anamnestic and diagnostic criteria.
beyond the nonspecific effects of nonpharmacotherapeutic origin
within the framework of a placebo-controlled trial that is universally
accepted.43,44 As such, the new drug must be tested against a placebo.43,44 Consequently, the ethical commission agreed with a placebo-controlled trial in the treatment of chronic (persistent) HE or
SHE.
The study was performed in accordance with the revised Helsinki
Declaration, the stipulations (sections 40-42) of the German Drug
Law, and the requirements of the EEC guidelines on ‘‘Good clinical
practice for trials on medicinal products in the European Community.’’ With respect to these standards, all patients had to declare
voluntary participation in this trial by signing an informed-consent
form. Further documentation included: the vote of the ethics commission of the Hesse State Medical Association of 15.01.1990, and the
registration of the clinical test according to section 67, paragraph 1,
of the German Drug Law.
Treatment Groups
A total of 126 cirrhotic patients fulfilled the criteria for inclusion
in this investigation performed between April 1990 and May 1991.
They were randomly assigned to receive placebo (n Å 63) or OA (n
Å 63). The two groups were similar in demographic characteristics,
etiology, duration, and severity of cirrhosis as determined by the
Child-Pugh criteria. (Table 1). In 12 patients, violations with respect
to the inclusion and exclusion criteria, to adverse events, adverse
drug reactions, or shortened duration of treatment occurred (for details, see Results). Four of these patients who had been taking prohibited medications and had been entered into the study erroneously
were retrospectively excluded for the treated-per-protocol analysis.
A fifth patient whose vision prevented him from performing the NCTA (inclusion criteria) was also withdrawn from the intent-to-treat
analysis even though he had received the full course of therapy.
The medication groups in the intent-to-treat sample as well as
those in the treated-per-protocol sample were homogeneous with regard to the anamnestic and diagnostic criteria. Discrepancies in the
composition of the two groups with respect to precipitant factors
could be minimized, because the patient population was well defined
by inclusion and exclusion criteria.
The primary analysis of the data on efficacy is based on the intent-
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FIG. 1. Effects of treatment with OA or placebo on performance time in NCT-A. Results (median, mean { SD) based on the evaluation of 126 cirrhotic
patients. Fifty percent of the observed NCT-A values are located within the 25th and 75th percentile (P 25 to P 75). The group differences between the
treatments were evaluated by means of the Wilcoxon two-sample test.
to-treat sample (total sample of 126 patients). In the intent-to-treat
analysis, changing samples at any time point were recorded due to
dropouts, missing data, and retrospectively excluded or withdrawn
patients. Treatment-per-protocol analysis was performed in 114 patients who showed no violation of inclusion or exclusion criteria and
no deviations of protocol. This analysis showed no changing samples.
Both statistical analyses showed virtually identical results. The data
presented here are based on the total study sample of 126 patients.
Subgroup Analysis. All 126 patients in the study were also retrospectively classified in subgroups (SHE, HE I, HE II) according to
the initial HE gradation (West-Haven criteria). Fifty-three of the
patients were assigned to the group of patients with initial SHE (OA
therapy: 26; placebo therapy: 27); 53 to the group with initial HE
classification I (OA therapy: 26; placebo therapy: 27); and 20 to the
group with initial HE classification II (OA therapy: 11; placebo therapy: 9). The need for and effectiveness of therapy was compared
and evaluated separately for this set of patients in the different
subgroups, according to the medication administered. Because the
original sample size calculation was directed to the whole study population, the sample size in each of the subgroups is relatively small
and might have introduced a type II error within the subgroup analysis.
RESULTS
Efficacy and Response to Therapy
NCT-A. NCT-A performance times on the first day of treatment (after 4 hours infusion and a superimposed protein load)
were similar in OA- and placebo-treated groups (58 { 23
seconds vs. 55 { 19 seconds) (Fig. 1). The mean decrease in
NCT-A score after 7 days was 19 { 19 seconds in the OA
group and 7 { 12 seconds in the placebo group. The comparisons of treatments showed significant group differences on
days 4 (P Å .0078) and 7 (P Å .0006) (Fig. 1) in favor of OA.
The NCT-A performance times increased as the HE changes
that existed initially increased in the individual subgroups
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of patients treated with OA or placebo (Table 2). After 7 days,
there were no longer any differences in final values attained
among the different subgroups treated with OA therapy. The
NCT-A times attained were in the vicinity of the normal for
noncirrhotic patients. In all of the three subgroups with an
initial HE gradation of SHE, HE I, HE II, the change of the
pre-/post-difference in the NCT-A showed descriptive statistical significances in the OA-treated patients (.001 õ P õ .01)
(Table 2) with the aid of the Wilcoxon signed rank test. Medication differences appear from comparison of the levels attained by the subgroups with OA therapy with those for the
corresponding placebo subgroups (Table 2). With respect to
the treatment-related pre-/post-differences in NCT-A, descriptive significances between OA and placebo could be
found in the subgroup of patients with initial SHE (P Å .0499)
as well as in HE I (P Å .0034). The most important pre-/postdifferences in NCT-A were found in the subgroups of patients
with initial mental state grade II (Table 2).
Venous Blood Ammonia Concentration. The pretreatment
fasting venous ammonia concentrations (mean { SD) were
similar in both treatment groups (OA: 81 { 38 mmol/L; placebo: 83 { 43 mmol/L) (Fig. 2). On average, the fasting venous
ammonia concentration decreased after 7 days by 17 { 37
mmol/L in the OA group and by 6 { 32 mmol/L in the placebo
group (pre-/post-differences). Significant differences between
the treatments in favor of OA (P õ .05, descriptive significance) were observed after 4 (P Å .0115) and 7 (P Å .0183)
days (Fig. 2).
The initial postprandial venous ammonia concentration on
the first day of therapy (i.e., after 4 hours of infusion and
12:00 PM protein load) was 82 { 37 mmol/L in the group
treated by OA versus 91 { 48 mmol/L in the group treated
by placebo. After 7 days of treatment, a mean reduction of
16 { 40 mmol/L in the OA group versus 10 { 36 mmol/L in
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TABLE 2. Comparison of the Effectiveness of OA with Placebo in the Treatment of Cirrhotic Patients with Respect to NCT-A,
NH4/ (Fasting/Postprandial) and PSEI in Consideration of the Initial HE Gradation (SHE, HE I, HE II)
OA
NCT-A
/
4
NH (fasting)
NH4/
(postprandial)
PSEI
SHE
N { SD
HE I
N { SD
HE II
N { SD
SHE
N { SD
HE I
N { SD
HE II
N { SD
SHE
N { SD
HE I
N { SD
HE II
N { SD
SHE
N { SD
HE I
N { SD
HE II
N { SD
Placebo
Initial
7 Days
Pre-/Post
Difference
Initial
7 Days
Pre-/Post
Difference
25
51.16 { 19.44
25
60.84 { 27.31
11
65.27 { 17.41
26
76.73 { 47.77
26
82.12 { 27.12
11
90.73 { 34.35
22
36.32 { 15.67
25
37.48 { 19.63
9
32.44 { 6.80
23
69.87 { 50.88
25
59.20 { 26.35
9
48.33 { 22.90
22
12.27 { 13.74‡
24
20.38 { 20.90‡
9
31.33 { 18.84†
23
5.22 { 32.94
25
22.76 { 40.91†
9
32.78 { 30.23*
27
46.59 { 16.17
27
56.00 { 15.02
9
74.78 { 21.83
26
73.00 { 27.94
27
93.81 { 57.61§
9
81.56 { 19.58
27
41.15 { 19.94
27
51.48 { 21.29
8
51.38 { 17.32
27
67.07 { 29.25
26
87.62 { 55.14§
8
75.25 { 31.29
27
5.44 { 11.72*
27
4.52 { 12.62
8
17.63 { 8.25*
26
5.12 { 36.57
26
7.50 { 31.05
8
7.13 { 25.85
26
69.65 { 38.73
25
88.68 { 31.81
11
97.00 { 35.51
25
0.185 { 0.065
24
0.296 { 0.062
11
0.468 { 0.064
23
72.04 { 52.96
25
64.92 { 29.86
9
54.44 { 24.19
22
0.110 { 0.107
25
0.146 { 0.101
9
0.164 { 0.055
23
0.174 { 37.00
24
24.5 { 39.44†
9
34.11 { 39.44*
22
0.076 { 0.091‡
23
0.140 { 0.119‡
9
0.286 { 0.088†
27
78.78 { 34.28
27
102.56 { 60.10#
9
92.78 { 34.20
27
0.184 { 0.059
27
0.302 { 0.073
9
0.472 { 0.067
27
68.30 { 37.71
26
93.27 { 60.72#
8
85.88 { 28.63
27
0.144 { 0.095
26
0.247 { 0.127
8
0.363 { 0.169
27
10.48 { 34.91
26
9.35 { 41.98
8
6.75 { 22.40
27
0.040 { 0.092*
26
0.055 { 0.097†
8
0.100 { 0.136
The table includes the respective number of investigated patients (N) as well as the mean level ({SD) of each subgroup during the course of this 7-day
treatment. The high SD in the NH4/ data in the subgroup of patients with initial HE I treated with placebo results from one patient (patient 19) with an
extremely high fasting (§ day 0: 317 mmol/L; day 7: 254 mmol/L) as well as postprandial (# day 0: 274 mmol/L; day 7: 147 mmol/L) NH4/ level. The Wilcoxon
signed rank test was used for testing descriptive statistical significances (*P ° .05; †P ° .01; ‡P ° .001) for the pre-/post-differences between the initial level
and the level after 7 days of treatment within each medication group. To find a difference between the medication groups (OA vs. placebo) with respect to the
pre-/post-differences, the Wilcoxon-Mann-Whitney U test (Wilcoxon two-sample test) was applied.
the placebo group could be seen. In a comparison of the final
values after 2 (P Å .012) and 4 (P Å .013) days of therapy,
significant differences between both groups in favor of OA
were observed after 2 and 4 days of therapy (P õ .013). On
day 7, a tendency toward statistical significance (P õ .078)
was shown (Fig. 2). Because of differences in the initial postprandial ammonia levels, the comparison of the pre-/postdifferences between the treatments by means of the Wilcoxon
two-sample test did not achieve significant effects (day 2: P
Å .053; day 4: P Å .065).
The pretreatment fasting and postprandial NH4/ levels in
the individual subgroups increased with the severity of the
initially existing HE in both medication groups (Table 2). The
initial data for the NH4/ values for the subgroups with HE
I and HE II, respectively, for the placebo group, which apparently do not agree with this postulate, are caused by the
extreme value of one patient with an initially determined HE
I (Table 2). The most effective ammonia reduction occurred
in the OA-treated subgroup of patients with HE grade II,
which initially showed the highest ammonia changes (HE II/
NH4//[fasting]/OA/initial: 90.7 { 34.4 mmol/L; day 7: 48.3 {
22.9 mmol/L; pre-/post-difference: 32.8 { 30.2 mmol/L; HE II/
NH4//[postprandial]/OA/initial: 97.00 { 35.31 mmol/L; day 7:
54.44 { 24.19 mmol/L; pre-/post-difference: 34.1 { 39.4 mmol/
L). There are also distinct differences between the final values attained when placebo therapy is compared with 7 days
of treatment with OA for subgroups HE I and HE II, respectively (Table 2). There are no detectable changes when comparing the two medication groups in the subgroups with initially existing SHE (Table 2).
Mental State. For better comparison to other published
data, the mean values of the treatment groups are provided
first. On day 0, both treatment groups gave comparative re-
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sults: in the placebo group, the mean mental state grade was
0.91 { 0.48 versus 0.97 { 0.53 in the OA group. During
the 7 days of placebo administration, the mean mental state
improved, but the mean scores after 2, 4, and 7 days of placebo treatment showed only minor differences from basal
(day 0) levels: 0.86 { 0.45, 0.82 { 0.47, and 0.72 { 0.52.
Mean scores in the OA-treated group fell progressively and
to a higher degree than in the placebo group. The mean values on days 2, 4, and 7 were 0.81 { 0.46, 0.64 { 0.42, and
0.42 { 0.33, respectively.
On the basis of frequencies of change in the mental state
grade, which may be more appropriate indicators for a rank
scale such as HE grading, a clear difference between OA and
placebo was observed (P õ .001, descriptive significance).
Improvement in the mental state grade was seen more frequently in the OA group than in the placebo group (59% vs.
32%). The most important effects can be seen in the subgroup
of patients with initially overt HE (grade I and II) (Table
3). Additionally, the ratio of improved versus not improved
mental state grade as well as the odds ratios for the subgroups with initial manifest HE (mental state grade I or II)
(Table 3) were calculated. As can be seen in Table 3, there
are differences in both odds ratios between the subgroups.
The test for homogeneity of the odds ratios was not significant
(P Å .2193), which indicates that both subgroups share an
odds ratio of 4.78 (95% confidence interval: 1.76; 36.08). In
the two-sided test, the common odds ratio is significantly
different by 1 (P Å .0026 [RGB variance] or P Å .0016 [M-H
variance]).
Thirty-seven OA-treated patients manifested a reduction
by at least one grade in mental state during the treatment
period; 26 patients showed no improvement. Seventeen of the
latter (65%) had SHE during the whole treatment period.
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HEPATOLOGY June 1997
FIG. 2. Effects of treatment with
OA or placebo on fasting, as well as
postprandial venous blood ammonia
concentrations. Results based on the
evaluation of 126 cirrhotic patients
(median, mean { SD) participating
in this 7-day, double-blind, placebocontrolled clinical trial. P 25 and P 75
represent the 25th and 75th percentile of an empiric distribution. Fifty
percent of the observed values are
within this range. The group differences between the treatments were
calculated on the basis of the pre-/
post-differences (Wilcoxon two-sample test). The pre-/post-differences
within each group were compared by
means of the Wilcoxon signed rank
test.
Only 9 of these 26 patients with initial SHE (35%) showed
improvement, which is defined as normalization of the initially prolonged NCT-A test time below the 30-second borderline in the case of mental state grade 0 after 7 days of OA
treatment. After 7 days of treatment, more than 76% of the
OA-treated patients with initial grade I and II exhibited improvement. Seventy-five percent of these patients had no HE
TABLE 3. Results in the Patient Subgroups
Improved
Not
Improved
Total
n (%)
n
n
9 (82)
2 (22)
11
2
7
9
11
9
20
4.50
0.286
15.75
19 (73)
12 (44)
31
7
15
22
26
27
53
2.71
0.80
3.39
Medication
Initial HE grade II
OA
Placebo
Total
Initial HE grade I
OA
Placebo
Total
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or only SHE (mental state grade 0; NCT-A time ú 30 seconds)
at the end of the OA treatment period. In contrast, 20 of
the placebo-treated patients showed improvement in mental
state grade compared with 43 patients who showed no response. Of these 43 patients, 21 had originally exhibited
SHE. Six of the 27 placebo-treated patients who had SHE
initially showed improvement (22%).
PSEI. On day 1, both groups (OA- and placebo-treated)
showed the same PSEI score (mean { SD: 0.28 { 0.12; N Å
60 [OA], N Å 63 [placebo]), which, in this trial, includes the
three estimated, above-mentioned parameters, namely mental state, NCT-A performance time, and postprandial blood
ammonia concentration based on the West-Haven criteria.9,11,40 At the end of therapy, a mean reduction of 0.14 {
0.13 (day 7: 0.135 { 0.10, N Å 56) was observed in the OA
group versus 0.05 { 0.1 (day 7: 0.22 { 0.14; N Å 61) in the
placebo group. The comparison of both treatments on day 7
(P Å .0011) as well as the comparison of the pre-/post-differences in OA and placebo-groups (P Å .0003) indicated a considerable difference in favor of OA (Wilcoxon two-sample
test). The PSEI reached after OA treatment was closer to
normal than after placebo (P õ .01 after 4 and 7 days).
The initial scores in the PSEI also increased in the individ-
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KIRCHEIS ET AL.
1357
FIG. 3. Comparison of the effectiveness of OA with placebo treatment in consideration of the initial HE gradation (SHE, HE I, HE II). The changes of the
mean levels of the PSEI of the respective subgroups are presented during the course of this 7-day treatment. The presentation of the upper and lower 95%
confidence limit of the mean (CLM) for each time point allows the conclusion of descriptive, statistically significant differences in or between the subgroups.
The subgroup differences between the treatments were calculated on the basis of the pre-/post-differences (Wilcoxon two-sample test). Medication differences
appear in the subgroups with initially manifest HE grade I (P õ .0153) and HE grade II (P õ .02) in favor of OA. No statistical group differences could be
proven between the subgroups of patients with initial SHE.
ual subgroups as the initially existing HE changes increased
in both medication groups (Table 2, Fig. 3). There are no
significant differences between the initial values for the particular medication groups (OA and placebo) when the matching subgroups (SHE, HE I, HE II) are compared. No differences can be detected between the final values (mean { SD)
of the scores (0.11 { 0.11 [SHE] to 0.16 { 0.06 [HE grade
II]) for the different subgroups after 7 days of OA therapy
(Table 2, Fig. 3). This tendency cannot be detected in the
subgroups treated with placebo (Fig. 3). Medication differences with respect to the pre-/post-differences after 7 days
of treatment appear in a comparison of the subgroups with
initially manifest HE grade I and II treated with OA (P õ .05)
and those treated with placebo (Table 2, Fig. 3). No statistical
group differences (Wilcoxon two-sample test) could be proven
between the subgroup of patients with initial SHE.
Adverse Events and Adverse Drug Reactions
OA was well tolerated in 86% of the treated patients. Placebo was tolerated well in 100% of the patients. Seven patients showed adverse events or adverse drug reactions and
a shortened treatment period. Two of these patients were
treated for 4 days. None of the other five had received treatment for longer than 2 days. Six of these seven patients had
received OA. Three had exhibited upper gastrointestinal and
central nervous symptoms such as nausea and/or vomiting
and were withdrawn from the study (5%). In none of the 63
patients in the placebo group were nausea and/or vomiting
observed. One patient treated with OA developed an acute
abdomen, secondary to a penetrating ulcer, and was withdrawn. One cirrhotic patient developed hepatorenal syndrome and was withdrawn. One patient refused to participate
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in the study and was withdrawn before therapy started. One
of the seven patients in the placebo group developed progressive heart failure and was withdrawn from the study after
the study medication had been started.
DISCUSSION
The treatment with OA, a stable salt of the natural Lamino acids ornithine and aspartic acid, results in significant
reductions in blood ammonia levels. This ammonia-lowering
effect of OA and other ornithine or aspartate conjugates has
been known for many years from both animal experiments
and clinical observations. Clinical findings were mainly
based on the results obtained in uncontrolled studies.24-26
Therefore, the present study was aimed at quantifying the
therapeutic efficacy of intravenously administered OA in a
randomized, placebo-controlled, double-blind trial in a defined patient population of cirrhotic patients with hyperammonemia and chronic (persistent) manifest HE and SHE.
Although we refrained from including patients with severe
HE, and despite the relatively short 1-week treatment, a
clear clinical benefit of OA was observed. This statement is
based on results proven within the whole study population
included as required by the inclusion and exclusion criteria,
as well as within the subgroup analyses. Improvements occurred using OA in both postprandial and fasting venous
ammonia levels compared with placebo. The postprandial
ammonia level estimated at 1:00 PM represents the reduction
of the postprandial hyperammonemia, induced by the protein
intake of 0.5 g protein/kg body weight of the midday meal,
after a 4-hour OA or placebo infusion in the morning (8:00
AM to 12:00 PM). It therefore represents the net effects of 4
hours of infusion on hyperammonemia in combination with
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HEPATOLOGY June 1997
the previous 2 to 7 days of OA infusion. The fasting ammonia
level estimated at 8:00 AM represents the reduction of the
fasting ammonia level after 2, 4, and 7 days after daily 4hour OA or placebo infusion, in contrast to the uninfluenced
baseline level, as a result of the OA- or placebo-induced effects. It therefore represents the impact of the previous 2 to
7 days of OA infusion.
It should be borne in mind that the relevant initial postprandial ammonia levels used to calculate the difference between OA and placebo was determined in patients undergoing OA or placebo treatment. In a previous study,27 it was
established that, at an identical protein intake of 0.50 g/kg
protein with the midday meal, a significant ammonia-reducing effect occurs during a 4-hour infusion of 20 g as well
as 40 g OA. Therefore, it can be assumed that the initial
postprandial ammonia levels on day 0 in the OA-treated patients had already been therapeutically influenced by the
intravenously administered OA. This is reflected by the lower
postprandial ammonia levels in the OA group (mean: 82 {
37 mmol/L) as compared with the placebo group (mean: 91 {
48 mmol/L). Because the baseline data in the fasting ammonia
levels confirm the comparability of both groups, ammonia
reduction in the OA group may be considerably greater than
that reflected by the present data. The significant effect of
OA on fasting ammonia levels confirms this conclusion.
A significant therapeutic benefit was also observed in favor
of OA in the NCT-A, which is an accepted and reliable psychometric test for the assessment of mental function in cirrhotic
patients with HE.9,11,40 Psychometric tests were performed at
the same time as the postprandial ammonia measurement
and the evaluation of the patient’s mental state. The aim was
to evaluate if the impact of the previous 4-hour infusion on
the protein-induced (12:00 PM meal [0.5 protein/kg body
weight]) hyperammonemia in combination with the previous
2 to 7 days of infusion resulted in improvement of NCT-A
and mental state grading. With regard to the whole study
population, a clear treatment effect in favor of OA could be
shown regarding improvement in NCT-A test time. Accordingly, a superiority of OA in comparison with placebo was
shown in the subgroups with manifest HE (HE I, HE II).
Interestingly, the evaluation of the patients with initial SHE
characterized by a prolonged NCT-A test time (ú30 seconds)
in the absence of manifest HE (grade 0) shows the only significant treatment-related effect in favor of OA in this subgroup
population with regard to improvement in NCT-A test time.
Otherwise, it could be shown that the response to OA in
patients with initial SHE is smaller than in those patients
with initially manifest HE (mental state grade I or II).
Decreases in mean mental state grading occurred twice as
frequently in the OA group as in the placebo-treated patients.
These findings show that OA administration approximates
the therapeutic results obtained with lactulose/lactitol and
neomycin.10-13 On the other hand, no improvement in mental
state grade was observed in 26 OA-treated patients, 17 of
whom (65%) had SHE at the beginning of treatment. The
separate evaluation of these patients with SHE also shows,
with regard to mental state gradation, that the response to
OA is smaller than in those patients with initial manifest
HE (mental state grade I or II). Based on the odds ratios, it
was observed that the greater the initial mental state gradation, the greater the effect of OA. These results indicate at
least for the subgroups of patients with initial manifest HE
a significant improvement with regard to their mental state
gradation. We would anticipate on the basis of the relationship shown by the odds ratio that patients with mental state
grades 3 and 4 would respond even more dramatically. It is
probable that, in the case of initial SHE, the responder rate
with regard to HE gradation will improve with further continuation or higher dose of OA treatment.
In the individual subgroups of patients with SHE, HE I,
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and HE II, it was found that the deviation from the normal
clearly increased as the initially existing mental state became
more pronounced, with respect to the changes detected in the
NCT-A, the changes in the NH4/ balance, and the changes
in the PSEI. Thus, the worst initial conditions occurred in
the groups with the greatest limitations of mental function.
No differences among the different subgroups could be detected in the final values attained after 7 days of therapy
with OA, whether for NCT-A, the NH4/ values (postprandial,
fasting), or the PSEI. The conformity of the improvements
with respect to the final values attained in the OA group
during the 7-day therapy indicates the efficacy of the OA
therapy in all the subgroups. Regardless of the severity of
the initial condition in these mild forms of HE, it is possible
with the OA therapy to reach a state—with respect to the
mental condition (HE gradation), the results in the psychometric tests (NCT-A), and the ammonium (NH4/)—that is
near or in the vicinity of the normal for all the subgroups.
Distinct differences from the placebo therapy can be shown
here. The improvements in the placebo groups are indeed
small, on the order of 10% to 15% spontaneous improvement
rates, but they are clearly different from the improvements
attained under OA. The more pronounced the initial changes
in HE gradation, NCT-A, or ammonium, the more pronounced are the reactions to effective therapy in this subgroup. The smaller the initial changes in HE gradation, NCTA, or ammonium, the more difficult it is to show an effective
therapeutic treatment in this subgroup. Because of this, no
significant improvements in comparison with placebo could
be shown regarding mental state gradation and ammonialowering in patients with initially existing SHE.
Another reason is due to a possible type II error that arises
from the sample size calculation, which, as planned, was
based on the whole study population. That also applies to
specific analyses in the subgroup of patients with initial mental state grade II. Because our definition of SHE was based
on the 30-second borderline in NCT-A, we cannot exclude an
erroneous diagnosis of SHE in a number of patients. At the
same time, the evidence of treatment differences because of
erroneously included patients could have been more difficult.
It is also clear from the results that SHE and overt HE have
different significance with respect to the necessity of the
treatment, but not with respect to its efficacy.
Adverse effects were not observed in patients in the placebo
group and in only 5% (3 patients) in the OA group. None
of the OA-induced side-effects were severe. These findings
confirm the results of uncontrolled open trials with OA in
over 4,500 patients that show that it is a safe, well-tolerated
therapeutic agent.
This investigation confirms the beneficial results of intravenous OA in several previous clinical trials. Henglein-Ottermann showed improvement in ammonia levels in a doubleblind crossover comparison of placebo and OA in normal
subjects and cirrhotic patients with hyperammonemia induced by infusions of ammonium chloride.26 They found that
5 g OA given over 1 hour caused a rapid decrease in blood
ammonia. Holm et al. studied various doses of OA infused
over 8 hours in cirrhotic patients with hyperammonemia induced by ingestion of a liquid protein diet.27 A single 8-hour
infusion of OA given in doses of 5 and 20 g diminished the
hyperammonemia in a dose-related manner. Forty grams of
OA completely abolished the increase in blood ammonia concentration. In addition, this large dose of OA increased the
branched chain amino acid–aromatic amino acid ratio.27
The mechanism by which ornithine-aspartate works is of
much theoretical and practical interest. With respect to the
proven functional heterogeneity of the hepatocytes,15,16,31-33
evidence was presented showing that glutamate, aspartate,
a-oxoglutarate, and citric dicarboxylates such as malate and
benzoate were taken up almost exclusively by perivenous he-
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KIRCHEIS ET AL.
patocytes.34-36 It was postulated that, under a pathological
condition such as cirrhosis, aspartate and also dicarboxylates
may serve as a carbon source and are consistent as a determinant for the impaired GS flux in the perivenous scavenger
cells.34-36 As has been shown, the flux through the impaired
urea cycle enzyme system, especially through carbamylphosphate synthetase, is increased by the administration of ornithine, taken up by the periportal hepatocytes.20-23
In contrast to hepatic GS, recent findings suggest fundamentally different regulatory mechanisms for GS and the
removal of excess ammonia in muscle and brain. Being devoid
of an effective urea cycle,30 these tissues rely on glutamine
synthesis via GS for the removal of excess ammonia. Whereas
GS activities in brain are decreased by portocaval shunting,45
enzyme activities in homogenates of skeletal muscle were
found to be significantly increased after shunt surgery.30 It
is conceivable, as reported by a previous study, that the addition of branched-chain amino acids to skeletal muscle homogenates resulted in further significantly increased GS activities in muscle tissue.37
Recent evidence suggests that ornithine may act in the
central nervous system to ameliorate HE in animal models.38
In addition to the stimulation of the periportal urea synthesis
in the liver, the proven increased transport of ornithine
across the blood-brain barrier indicates the central nervous
system as a second target for ornithine. Both mechanisms
and targets afford reasonable explanations for the reduction
of increased blood ammonia concentration by ornithine associated with lower concentrations of glutamine and lactate in
brain.39 These changes may be involved in the prevention of
brain edema and may explain the simultaneous improvement
of HE gradation shown by ornithine administration in these
models.39 Further clinical and experimental investigations on
the role of skeletal muscle and brain in the possible mechanisms of beneficial action of OA in HE are in progress.
In summary, OA infusion resulted in significant, improved
NCT-A times, mental state grading, and PSEI scores, as well
as in decreased fasting and postprandial venous ammonia
concentrations compared with placebo. In subgroups retrospectively classified according to their initial HE gradation
(SHE, mental state gradation I and II), OA showed descriptive significances in the subgroups with manifest HE with
respect to ammonia-lowering, improvement in NCT times,
and mental state gradation. Subgroup analyses in patients
with initial SHE revealed medication differences in the psychometric test used in favor of OA. Consistently, OA appears
to be a safe and effective treatment of chronic (persistent)
manifest HE. Based on the results seen in the subgroup of
patients with SHE, it is fair to assume that an effect of OA
will be proven if the type II error could be excluded and more
sensitive diagnostic criteria could be used.
All studies of OA to date were performed in patients with
SHE or mild, manifest HE (mental state grade I or II). Additional investigations in patients with SHE as well as with
more severe HE (grades III and IV) are in progress.
Acknowledgment: The authors thank the physicians and
staff of all the author-affiliated hospitals and laboratories for
their cooperation in the performance of the trial and for making their clinical and laboratory data available to us. The
authors gratefully acknowledge the secretarial assistance of
Ms. Annette Dreher.
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