Alcohol & Alcoholism Vol. 41, No. 4, pp. 372–378, 2006 doi:10.1093/alcalc/agl032
Advance Access publication 10 May 2006
ARGININE CHALLENGE UNRAVELS PERSISTENT DISTURBANCES OF UREA CYCLE AND
GLUCONEOGENESIS IN ABSTINENT ALCOHOLICS
MARTIN HASSELBLATT1, HENNING KRAMPE1, SILKE JACOBS1, HEIKE SINDRAM1,
VICTOR W. ARMSTRONG2, MARKUS HECKER3 and HANNELORE EHRENREICH1*
Division of Clinical Neuroscience, Max-Planck-Institute of Experimental Medicine, 2Department of Clinical Chemistry, Georg-August-University,
Gottingen, Germany and 3Institute of Physiology and Pathophysiology, Ruprecht-Karls-University, Heidelberg, Germany
(Received 27 October 2005; first review notified 9 January 2006; in revised form 22 March 2006; accepted 23 March 2006;
advance access publication 10 May 2006)
Abstract — Aims: Data on recovery from hormonal and metabolic sequelae of alcoholism in strictly controlled alcohol abstinence are
mainly restricted to short-term abstention. Our previous findings of persistently decreased plasma and urinary urea concentrations in
long-term abstinent alcoholics prompted us to further elucidate this unexplained phenomenon. Methods: The response of circulating
urea cycle metabolites and glucose-regulating hormones to an intravenous load (30 g) of arginine hydrochloride was investigated in
abstinent male alcoholics (n = 14) after complete recovery of all routine liver parameters and compared with that in healthy male con-
trols (n = 15). Results: The arginine challenge provoked (i) higher peak concentrations of arginine and increased arginine/ornithine and
ornithine/citrulline ratios in the plasma of abstinent alcoholics; (ii) augmented plasma glutamine concentrations in alcoholics in the
presence of comparable levels in both experimental groups of plasma glutamate, ammonia, and nitrate/nitrite; (iii) parallel increases
in plasma urea concentrations over the respective baseline levels but distinctly higher urinary urea excretion in controls; (iv) a blunted
blood glucose response to arginine in alcoholics together with a reduced insulin and glucagon surge; and (v) an elevated growth hor-
mone peak as compared with controls. Conclusions: Application of an intravenous arginine challenge reveals profound and lasting
metabolic and hormonal disturbances in abstinent alcoholics, affecting urea cycle and gluconeogenesis. The common denominator of
many of these changes may be an acquired irreversible deficiency in cellular energy regulation.
INTRODUCTION synthetase, ornithine transcarbamoylase, argininosuccinate
synthetase, and argininosuccinase. The formation of car-
Alcohol dependence is an as yet incurable human brain bamoylphosphate by carbamoylphosphate synthetase and of
disease with the molecular mechanisms of irreversibility still argininosuccinate by argininosuccinate synthetase are the
widely unknown. In fact, alcoholism causes a variety of energy-dependent steps. The amount of arginine utilized for
profound and lasting hormonal and metabolic disturbances ureagenesis depends on arginase activity (Morris, 1992) and
but data on their recovery in strictly controlled alcohol regulates the activation of carbamoylphosphate synthetase by
abstinence remain scarce. Careful follow-up of the long-term N-acetyl-glutamate (Shigesada and Tatibana, 1971).
regeneration of peripherally accessible parameters of organ The present study was designed as a first step to uncover
function, however, may shed some light on potential deter- mechanisms underlying the persistently decreased urea levels
minants of irreversibility also in the brain. While liver enzyme in abstinent alcoholics. Using intravenous arginine as a sub-
activities in plasma normalize within weeks of abstention strate load for the urea cycle, the capacity of urea synthesis
from drinking, other parameters such as erythrocyte mean in vivo was challenged in abstinent alcoholics and controls.
corpuscular volume take months to years to recover In our setting, arginine infusion, a procedure commonly and
(Hasselblatt et al., 2001). Regulatory hormonal circuits, e.g. safely applied in diagnostic endocrinology (Ghigo et al.,
the hypothalamic-pituitary-adrenal or the hypothalamic- 2001), should unmask (i) a potentially altered rate of synthesis
pituitary-gonadal axis, remain impaired for months of urea cycle components, i.e. ornithine, citrulline, and
(Ehrenreich et al., 1997b; Hasselblatt et al., 2003). Low argininosuccinate, (ii) a potential shift of arginine and ammo-
plasma urea concentrations have been reported in drinking nia metabolism towards alternative pathways of nitrogen
and early abstinent alcoholics (Stamm et al., 1984; removal including formation of glutamine and nitric oxide,
Wallerstedt and Olsson, 1978). In previous work we made and (iii) disturbed response patterns of glucose-regulating
the remarkable observation that the decreased plasma and hormones stimulated by arginine (Merimee et al., 1965;
urinary urea concentrations in alcoholics persist even over Floyd et al., 1966).
months and years of strictly controlled abstinence, suggesting
sustained disturbances of urea synthesis within the urea cycle EXPERIMENTAL SUBJECTS
(Doring et al., 2003; Jahn et al., 2004).
In humans, the urea cycle is essential for the elimination of After approval of the study by the Committee for Medical
ammonia, a by-product of protein catabolism and utilization of ¨
Ethics of the Georg-August-University, Gottingen, Germany,
most amino acids for gluconeogenesis. The immediate pre- informed consent for participation was obtained from 15
cursor of urea is arginine, which is hydrolysed to urea and healthy male controls and 14 male alcoholics.
ornithine by arginase. Arginine in turn is synthesized by the After inpatient detoxification of 1–2 weeks, patients joined
first four enzymes of the urea cycle, i.e. carbamoylphosphate the OLITA (Outpatient Longterm Intensive Therapy for Alco-
holics) programme, where abstinence was ascertained through
daily contacts (including weekends and holidays) and daily
*Author to whom correspondence should be addressed at: Tel.: +49 551 3899 urine analyses for alcohol (Ehrenreich et al., 1997a). Median
628; Fax: +49 551 3899 670; E-mail: email@example.com age of the subjects was 40 years (range, 34–53). Their median
Ó The Author 2006. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved
ARGININE CHALLENGE IN ABSTINENT ALCOHOLICS 373
body mass index (BMI) was 22.8 kg/m2 (range, 18.4–29.4). Table 1. Assay data
Subjects had a history of 9 (median) (range, 3–24) years of CV intra-assay (%) CV inter-assay (%) Detection limit
alcohol dependence according to DSM IV criteria with a
median daily consumption of 317 g (range, 120–960) of pure C-Peptide 5 6 5 pmol/l
GLP-1 9 6 2 pmol/l
alcohol over the last half year before entering the programme. Glucagon 7 10 20 ng/l
Subjects displayed no signs of liver cirrhosis as confirmed Growth hormone 2 14 0.5 mIU/L
by physical and laboratory examination, and abdominal ultra- Insulin 4 5 0.5 mU/l
sound imaging, were free of other severe or chronic illnesses
and had no history of drug abuse (except for alcohol, nicotine,
and caffeine). They had received clomethiazole, magnesium, Table 2. Baseline laboratory values: liver enzyme activities, total protein,
potassium, and vitamin B1 during the first week of abstinence. albumin and urea in abstinent alcoholics and controls (mean ± SD)
Starting from the second week, 50 mg oral calcium carbimide Alcoholics (N = 14) Controls (N = 15) P-value
(Dipsan) was given as aversive medication daily over the first
ALAT (U/l) 10 ± 6 15 ± 5 n.s.
3 months of abstinence. From the fourth month on, patients ASAT (U/l) 10 ± 3 10 ± 3 n.s.
were switched to disulfiram (Antabuse), 3 · 400 mg/week gGT (U/l) 13 ± 6 12 ± 4 n.s.
(Ehrenreich et al., 1997a). Although there is no indication Cholinesterase (U/l) 6670 ± 813 6749 ± 907 n.s.
from literature that acetaldehyde dehydrogenase inhibitors Protein (g/l) 74 ± 5 75 ± 4 n.s.
Albumin (g/l) 42 ± 3 44 ± 2 n.s.
influence any of the parameters of interest here, this medica- Plasma urea (mmol/l) 3.8 ± 0.9 4.9 ± 1.1 P < 0.01
tion was stopped 2 weeks before the arginine challenge. The Urinary urea (g/24 h) 8.4 ± 2.3 11.7 ± 3.9 P < 0.05
control group comprised 15 healthy male volunteers without
evidence of alcohol or drug abuse. Controls were matched as
closely as possible to patients with regard to age (39 years,
range 30–59), BMI (24.9 kg/m2, range 18.7–31.8), and (ELISA; LINCO Research). Samples from patients and
cigarette consumption. controls were always assayed in random order. Data on
intra-assay and inter-assay variations are compiled in Table 1.
Plasma and urinary concentrations of nitrate/nitrite were
MATERIALS AND METHODS measured as described (Moshage et al., 1995). Blood glucose
concentrations were serially determined from capillary blood
Experimental procedures (Glucometer Elite, Bayer, Germany). Total protein, albumin,
ammonia, and urea were determined using standard laboratory
After 13 weeks (range, 8–19) of controlled alcohol abstinence,
techniques. Liver enzyme activities [alanine amino transa-
the arginine infusion test was performed after an overnight
minase (ALAT), aspartate amino transaminase (ASAT),
fast. At 7:00 h, subjects were placed in a comfortable supine
cholinesterase (CHE)] were determined at 37 C and were
position. Two intravenous catheters were inserted in both
calculated equivalent to a measurement at 25 C.
forearms and kept open with a slow infusion of 0.9% saline
for separate blood sampling and drug administration. Blood
pressure and heart rate were monitored continuously. At Data analysis
09:00 h, 30 g of L-arginine-hydrochloride (Braun, Melsungen, Data are presented as mean ± SD in the text and as mean ±
Germany) was infused intravenously over 30 min by means SEM in the figures. Statistical analysis was performed by
of an automated infusion pump (Braun, Germany). Venous Student’s t-test. For multiple comparisons between groups
blood samples were collected at time points 0 (start of and over time ANOVA was used. Post hoc testing was done
infusion), 15, 30, 45, 60, 90, 120, and 180 min. Urine was if appropriate by Duncan’s test applying the Statistica soft-
collected throughout the preceding 24 h (09:00 h–9:00 h), ware package (Statsoft Inc., Tulsa OK). Significance level
from 09:00 h–12:00 h during the test, and for the following was P 0.05.
24 h interval.
Analytical procedures RESULTS
Plasma for determination of amino acid and hormone concen- Baseline laboratory values
trations was collected in lithium heparin tubes, immediately
separated by centrifugation (2000 g, 10 min, 4 C), and stored By the time the arginine stimulation test was performed, i.e.
at –80 C until analysed. Plasma concentrations of the amino after a median of 13 weeks (range, 8–19) of strictly controlled
acids arginine, ornithine, citrulline, argininosuccinate, glutam- alcohol abstinence, plasma activities of liver enzymes had nor-
ine, and glutamate were measured by HPLC (Meyer et al., malized. Plasma concentrations of total protein and of albumin
1997). Except for hydroxyarginine (0.5 mmol/l), the detection were comparable with that of controls. Notably, plasma as
limit for all amino acids was 0.8 mmol/l. Plasma concentra- well as urinary urea concentrations were significantly lower
tions of insulin, c-peptide and growth hormone were measured in abstinent alcoholics (Table 2).
using commercially available immunoradiometric assays
(Immunotech, Prague, Czech Republic). Glucagon concentra- Blood pressure, heart rate, and adverse events upon
tions were measured by radioimmunoassay (LINCO Research, arginine administration
Missouri). Glucagon-like-peptide 1 (GLP-1) concentrations In both abstinent alcoholics and controls mean arterial pres-
were determined by enzyme-linked immunosorbent assay sure (range, 85–92 mmHg) and heart rate (range, 60–75/min)
374 M. HASSELBLATT et al.
did not change significantly during the arginine infusion test. (ANOVA P = 0.08; Fig. 4C). Glucagon concentrations
Adverse events were not encountered. increased transiently to peak at 0:30 h mirroring the insulin
response to arginine infusion. Again, the increase in plasma
Concentrations of plasma urea and urea cycle metabolites glucagon concentrations was significantly lower in alcoholics
upon arginine administration (219 ± 60 versus 312 ± 104 pg/ml, Fig. 4D). No significant
Baseline plasma arginine concentrations were low in both changes in plasma GLP-1 concentrations were observed over
alcoholics and healthy controls (67 ± 37 versus 61 ± time nor between groups (data not shown).
39 mmol/l, respectively). Intravenous administration of
arginine resulted in significant increases in plasma arginine Growth hormone concentrations upon arginine infusion
concentrations over time. In both alcoholics and controls, Plasma growth hormone concentrations increased transiently
plasma arginine concentrations peaked at 30 min, i.e. at the to peak at 1:00 h. In abstinent alcoholics, the increase in
end of the arginine infusion. Peak arginine concentrations growth hormone concentrations was significantly augmented
were significantly higher in alcoholics as compared to controls as compared with that in controls (1:00 h, 21.7 ± 28.9 versus
(3222 ± 771 versus 2344 ± 975 mmol). Whereas absolute 7.6 ± 11.7 mU/l) (Fig. 5).
increases in plasma urea concentrations in abstinent alcoholics
paralleled those of controls, the arginine/urea ratios were
much higher in alcoholics. Increases in plasma ornithine and
citrulline concentrations were observed in both groups. DISCUSSION
Ornithine concentrations peaked at 60 min and tended to be
higher in alcoholics versus controls, while peak citrulline The present study employed an intravenous arginine challenge
concentrations tended to be lower. The corresponding ratios, to unravel persistent alterations in circulating urea cycle com-
i.e. arginine/ornithine and ornithine/citrulline, were signifi- ponents and major metabolic hormones that clearly outlast the
cantly increased in abstinent alcoholics. Controls displayed normalization of plasma liver enzyme activities in long-term
higher baseline plasma argininosuccinate concentrations, abstinent alcoholics. In comparison with healthy controls,
which decreased over time to reach levels of abstinent alcohol- alcoholics started from lower plasma urea levels and demon-
ics. Ratios of citrulline/argininosuccinate increased, whereas strated in response to arginine: (i) higher peak concentrations
argininosuccinate/arginine ratios decreased over time. Both of arginine and increased arginine/ornithine and ornithine/
ratios did not differ significantly between abstinent alcoholics citrulline ratios; (ii) elevated plasma glutamine levels but
and controls (Fig. 1). unaffected plasma glutamate, ammonia, and nitrate/nitrite;
(iii) distinctly lower urinary urea excretion despite increases
Concentrations of ammonia, glutamine, glutamate, and in plasma urea concentrations over baseline; (iv) a blunted
nitric oxide metabolites upon arginine administration plasma glucose stimulation together with a reduced insulin
and glucagon surge; (v) an elevated growth hormone peak.
Baseline plasma ammonia concentrations were low in both Although several parameters measured in plasma only
alcoholics and healthy controls (1.9 ± 0.7 mmol/l versus indirectly reflect disturbances of intracellular metabolites,
1.8 ± 0.9 mmol/l, respectively) with no significant changes i.e. represent a spill-over into the extracellular space, the
over time or between groups (Figs 2 and 3). In contrast, discovery of such a globally altered pattern of metabolic and
plasma glutamine concentrations were significantly increased hormonal response to arginine in strictly abstinent alcoholics
in abstinent alcoholics as compared with that in controls at may encourage further research on the mechanisms of
0:30 and 1:00 h, whereas plasma glutamate concentrations irreversibility.
tended to be lower in alcoholics (Fig. 2A and B). No signific- Since administration of arginine resulted in comparable
ant changes in plasma nitrate/nitrite concentrations were increases in plasma urea over the respective baseline in both
observed over time or between groups (Fig. 2C). Plasma experimental groups, a reduced activity of arginase per se is
hydroxyarginine concentrations remained below the detection unlikely to fully account for the persistently lowered urea con-
limit in most abstinent alcoholics as well as in controls (data centrations in abstinent alcoholics. The increased arginine
not shown). Urine analysis revealed significantly lower total peak in alcoholics, in turn, could be explained by a delayed
excretion of urea and a tendency for decreased nitrate/nitrite cellular uptake of arginine, which has been described in rat
excretion in alcoholics upon arginine challenge (Fig. 3). liver cells upon ethanol exposure (Rosa and Rubin, 1980).
Similarly, a reduced capacity of the ornithine/citrulline-carrier
Concentrations of glucose and glucose-regulating hormones might cause a delayed transport of ornithine into the mito-
upon arginine administration chondrial compartment, contributing to an accumulation of
In healthy controls, arginine administration was accompanied ornithine and an increased ornithine/citrulline ratio. In any
by a prominent transient rise in glucose concentrations fol- case, the elevated plasma arginine concentrations along with
lowed by a decrease at later time points. This hyperglycaemic increased plasma arginine/ornithine and ornithine/citrulline
response to arginine was blunted in abstinent alcoholics ratios and decreased urinary urea excretion upon intravenous
(5.2 ± 0.9 versus 5.8 ± 0.7 mmol/l; Fig. 4A). Insulin and arginine challenge point towards an overall reduced capacity
C-peptide concentrations increased transiently to peak at of the urea cycle in abstinent alcoholics. How else can such
0:30 h. Again, the increase in insulin in response to arginine reduced capacity be explained?
was significantly lower in alcoholics as compared with that Arginine stimulates the activation of carbamoylphosphate
in controls (19 ± 11 versus 31 ± 19 mU/l; Fig. 4C). Similarly, synthetase by N-acetyl-glutamate (Shigesada and Tatibana,
C-peptide concentrations tended to be lower in alcoholics 1971). An intravenous arginine challenge would, therefore,
376 M. HASSELBLATT et al.
A 70 A ∗
Glutamate 60 6,0
00:00 00:30 01:00 01:30 02:00 5,0
1000 ∗∗ ∗
00:00 00:30 01:00 01:30 02:00 02:30 03:00
0 B 35 ∗∗
00:00 00:30 01:00 01:30 02:00
00:00 00:30 01:00 01:30 02:00
Fig. 2. Plasma concentrations of glutamate (A), glutamine (B), and nitrite/ 0:00 0:30 1:00 1:30 2:00 2:30 3:00
nitrate (C) upon arginine administration in abstinent alcoholics (n = 14, closed
circles) and healthy controls (n = 15, open circles) over time (h:min).
*P 0.05, **P 0.01.
0:00 0:30 1:00 1:30 2:00 2:30 3:00
-24:00-0:00 0:00-3:00 3:00-24:00 350 ∗∗
-24:00-0:00 0:00-3:00 3:00-24:00 0:00 0:30 1:00 1:30 2:00 2:30 3:00
Fig. 4. Capillary blood concentrations of glucose (A), and plasma concentra-
Fig. 3. Amount of urinary urea (A) and nitrate (B) in abstinent
tions of glucose-regulating hormones, insulin (B), C-peptide (C), and gluca-
alcoholics (n = 14, black bars) and healthy controls (n = 15, white
gon (D), upon arginine administration in abstinent alcoholics (n = 14, closed
bars) before, during and after intravenous arginine load. *P 0.05,
circles) and healthy controls (n = 15, open circles) over time (h:min).
*P 0.05, **P 0.01.
ARGININE CHALLENGE IN ABSTINENT ALCOHOLICS 377
be expected to increase the capacity of the urea cycle, result- 35
ing in enhanced turnover and, subsequently, rapid normaliza- ∗∗
Growth Hormone (mIU/L)
tion of plasma arginine and ornithine concentrations. The
finding of increased plasma arginine concentrations together ∗∗
with increased plasma arginine/ornithine and ornithine/citrul-
line ratios in abstinent alcoholics indirectly indicates a 20 ∗
decreased activity of carbamoylphosphate synthetase. Since
the generation of carbamoylphosphate is the first ATP- 15
dependent step within the urea cycle, such decreased activity
of carbamoylphosphate synthetase might best be explained 10
by a reduced availability of ATP. Indeed, chronic ethanol
exposure has been shown to result in depression of hepatic 5
mitochondrial energy metabolism causing impaired citrulline 0
and urea synthesis in rat hepatocyte mitochondria upon
0:00 0:30 1:00 1:30 2:00 2:30 3:00
ethanol exposure (Cunningham et al., 1990; Adachi et al.,
1995). Fig. 5. Growth hormone plasma concentrations upon arginine administration
Accordingly, our finding of significantly higher glutamine in abstinent alcoholics (n = 14, closed circles) and healthy controls (n = 15,
plasma concentrations in abstinent alcoholics following the open circles) over time (h:min). * P 0.05, ** P 0.01.
arginine challenge might reveal a compensatory shift towards
elimination of ammonia by glutamine synthetase, as has been ¨
and water balance (Doring et al., 2003). In fact, we have pre-
reported for chronic ethanol exposure in perivenous rat hep- viously shown lasting suppression of circulating vasopressin
atocytes (Garcia-Ruiz et al., 1994). Exhaustion of this reserve ¨
in long-term abstinent alcoholics (Doring et al., 2003). Vaso-
under pathophysiological conditions may predispose alcohol- pressin increases urea synthesis in isolated rat hepatocytes
ics, even after long-term abstinence, for cerebrotoxic con- by enhancing the rate of mitochondrial citrulline synthesis
sequences due to both elevated glutamine and ammonia (Corvera and Garcia-Sainz, 1982) and stimulates glutaminase
levels (Cooper, 2001). The latter were found to be unaltered activity (Corvera and Garcia-Sainz, 1982; Drew et al., 1985).
in our experimental setting, pointing to a still sufficient elim- To conclude, application of an intravenous arginine chal-
ination of ammonia. In addition, findings of unchanged lenge uncovers persistent metabolic and hormonal disturb-
plasma concentrations of nitrite/nitrate and hydroxyarginine ances in abstinent alcoholics, affecting the urea cycle and
in response to arginine argue against an increased conversion gluconeogenesis. The common denominator of many of these
of arginine to nitric oxide in abstinent alcoholics. Regarding changes appears to be an acquired irreversible deficiency in
the higher plasma glutamine levels in abstinent alcoholics cellular energy regulation. This deficiency may predispose
after arginine infusion, cortisol driven glutamine synthesis alcohol-dependent men, despite long-term abstinence, for a
and release cannot be entirely ruled out as a contributing more rapid decompensation of metabolic/protective cellular
mechanism. pathways. Similar mechanisms in the brain may underlie the
Under fasting conditions, arginine is utilized for the genera- phenomenon of dependence.
tion of glucose. The blunted glycaemic response of alcoholics
to intravenous arginine may point (along with the significantly Acknowledgements — We would like to thank Arnold Hasselblatt (Department
of Pharmacology, Georg-August-University, Gottingen) for his valuable
lower insulin and glucagon peaks) towards impaired gluco- comments.
neogenesis. Impaired gluconeogenesis, in turn, further reflects
the long-term disturbance of energy metabolism in abstinent
alcoholics. Indeed, our findings in alcoholics closely resemble
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