Mechanism of the alcohol cyclic pattern: role of catecholamines
Jun Li,
Barbara A. French,
Paul Fu,
Fawzia Bardag-Gorce, and
Samuel W. French
Department of Pathology, Harbor-University of California Los Angeles
Medical Center, Torrance, California 90509
Submitted 24 February 2002
; accepted in final form 8 April 2003
 |
ABSTRACT
|
---|
The cause of the urinary alcohol level (UAL) cycle in rats fed ethanol at a
constant rate has been shown to involve the hypothalamic-pituitary thyroid
axis. Because the effect of thyroid hormone on the metabolic rate is augmented
by catecholamines, the role of catecholamines was investigated by using the
intragastric ethanol feeding model of alcoholic liver disease in which the UAL
cycles over a 6- to 10-day period. The diet was supplemented with ephedrine
and caffeine to test the hypothesis that the UAL cycle involves
catecholamines. The UAL was followed to see whether the cycle was ablated by
catecholamine supplements. Ethanol fed alone increased the blood levels of
catecholamines significantly more than did ephedrine fed alone. However, blood
catecholamine levels were significantly higher when ethanol was fed with
ephedrine compared with the sum of ethanol and ephedrine fed alone. This
indicated that the effect of ethanol and ephedrine were synergistic. The UAL
cycle was completely ablated in the ethanol + ephedrine-fed rats. These rats
tolerated a much higher dose of ethanol, indicating that they metabolized
alcohol faster due to an increase in metabolic rate caused by ephedrine. In
the ethanol + ephedrine-fed rats the liver pathology included significantly
higher alanine amino transferase (ALT) in the blood and centrilobular ischemic
necrosis in the liver. Necrosis was not present in the rats fed ephedrine
alone. In conclusion, catecholamine supplements prevented the UAL cycle by
increasing the metabolic rate to the point at which fluctuations in the
metabolic rate caused by alcohol were prevented.
thyroxine; metabolic rate; hypoxia; liver necrosis
THE URINARY ALCOHOL CYCLE seen in the intragastric ethanol
feeding rat model of early alcoholic liver disease has been shown to be
thyroid hormone dependent (8).
The urinary alcohol level (UAL) cycle was prevented by propylthiouracil
treatment and pituitary stalk severance
(8) or by treatment with a high
dose of thyroxine (7). However,
a high dose of thyroxine given with ethanol led to centrilobular ischemic
necrosis (7). The necrosis was
thought to be due to a hypermetabolic state induced by the combination of
hyperthyroidism and high blood alcohol levels (BAL). Liver hypoxia was
suspected at high BAL at the peak of the UAL cycle because ATP levels were
reduced and the NADH/NAD ratio shifted to the reduced state at the peaks
(1). The idea that hypoxia
occurred at the UAL peaks was supported by the increase in hypoxic indicators
such as erythropoietin and VEGF upregulation and an increase in hypoxyprobe
labeling of hepatocytes measured by immunofluorescence
(1). Thyroxine treatment
prevented the UAL cycle by increasing the metabolic rate as was indicated by
the increased rate of ethanol elimination observed
(7).
To further investigate the mechanism involved in the UAL cycle and
centrilobular necrosis seen in the intragastric feeding model, rats were fed
ethanol together with the adrenergic drug ephedrine and caffeine as used in
weight loss dietary regimens
(24).
Similar dietary regimens have occasionally caused hepatic toxicity, but the
nature of the toxicity was not defined
(5). Adrenergic drugs are known
to increase the metabolic rate and thus accelerate the ethanol elimination
rate (9) as was observed during
the UAL cycle (10). Caffeine
enhances the adrenergic effect on metabolism, increasing cAMP levels induced
by adrenergic signaling. It does this by inhibiting the breakdown of cAMP by
phosphodiesterase. Thyroid hormone treatment also accelerates the ethanol
elimination rate (7). Thyroid
hormone potentiates the ability of catecholamines to increase the rate of
O2 consumption (3).
In the UAL cycle, a cyclic increase in the rate of O2 consumption
was observed despite a constant rate of ethanol feeding
(8). Thus the data are
consistent with the hypothesis that the combination of ethanol and
catecholamine feeding will, like thyroxine, accelerate the rate of ethanol
elimination and thus block the UAL cycle. The results of this report further
support the hypothesis, deeming it to be correct.
 |
MATERIALS AND METHODS
|
---|
Male Wistar rats weighing 300 g (Harlan Sprague Dawley, Hollister, CA) were
fed diet and ethanol intragastrically continuously 24 h/day for 6 wk together
with pair-fed controls fed dextrose isocalorical to ethanol (groups 3
and 4, respectively). Two other groups were fed the diet supplemented
with ephedrine (50
mg·kg-1·day-1) and
caffeine (175
mg·kg-1·day-1). One
of these groups was fed ethanol (group 1), and one was pair-fed
isocaloric dextrose (group 2). All four groups had five rats per
group. Ethanol was fed at a constant rate of 13
g·kg-1·day-1 at
which 40% of the total calories were derived from ethanol or isocaloric
dextrose. The diet fed was previously described
(8). Of total calories 28.9%
were derived from fat, 6.2% from dextrose, and 24.9% from protein.
UALs were measured daily over the terminal 20- to 25-day test period. Urine
was collected over a 24-h period. Urine was protected from evaporation by an
overlay of toluene. UAL was measured colorimetrically (QED Saliva Alcohol Test
A150; ST Technologists, Bethlehem, PA). Blood levels of serum alanine
aminotransferase were measured by using a clinical analyzer (a kinetic rate
method on Synch Roncx Systems; Beckman Instruments, Brea, CA). Total blood
catecholamines were measured by HPLC (Quest Diagnostics, San Juan Capistrano,
CA). Body weights were recorded weekly for 6 wk of feeding. Liver
weight-to-body weight ratios were calculated at the time of death.
During the terminal 20- to 25-days, the UAL were charted to determine
whether the UAL cycle was manifest and to determine the ethanol dose each rat
could eliminate beginning at day 15 of recording the UAL cycle. On
day 15, the dose of ethanol was increased serially every few days to
14, 15, 16, and 17
g·kg-1·day-1 to the
dose, which proved lethal to the rat because of overdose. In this way, the
dose of ethanol that could be eliminated by each rat was determined. At the
time of death or in the case of controls death, the liver tissue was fixed in
zinc formalin (10%) and processed for histological examination by using
hematoxylin and eosin and reticulin staining.
The research protocol was approved by the Research and Education Institute
Animal Care Committee in accordance with the guidelines for animal care as
described by the National Academy of Sciences (1996).
Statistical methods employed were ANOVA and all pairwise multiple group
comparisons (Student-Newman-Keuls method).
 |
RESULTS
|
---|
Weight gain or loss in rats from the four experimental groups is shown in
Fig. 1. Note that the rats fed
ethanol or pair-fed dextrose gained weight, whereas the rats fed ephedrine and
caffeine in their diet lost weight. This was despite the fact that they
received the same amount of calories over 24 h as the pair-fed rats. The
weight loss was even greater when the rats were fed ephedrine + caffeine +
ethanol compared with rats pair-fed ephedrine + caffeine + dextrose
(P < 0.001) (Figs.
1,
2). The change in body weight
over the 6-wk feeding period was significantly different
(Fig. 2) (P <
0.001). The starting weight for all four groups was not different (Figs.
1,
2).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2. Body weights for all 4 experimental groups are given as starting and ending
weights (means ± SE, n = 5). Note that there are statistically
significant differences between start and end body weights of all groups as
well as among groups as indicated.
|
|
End body weights for the treated groups 1 and 2 were
statistically different when compared with the untreated groups (P
< 0.001) (Fig. 2). These
differences establish the weight loss effect of the ephedrine and caffeine
diet treatment when the rats were pair-fed, indicating that catecholamine
treatment increased the metabolic rate in the ephedrine and caffeine treated
rats. Ethanol + ephedrine + caffeine further increased the weight loss
presumably because the blood catecholamines were high in this group.
The liver weight-to-body weight ratio was calculated as a measure of
relative liver enlargement (Fig.
3).

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3. Liver weight/body weight ratios for all 4 experimental groups are given
(means ± SE, n = 5). Note that the treatment given with
ethanol feeding significantly increased the ratio compared with the other 3
groups as indicated. The ratio of liver weight/body weight increased in
group 1 despite the absence of liver fatty change partly due to a
greater decrease in body weight (Fig.
4).
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4. Liver weight of all 4 groups of rats. Note that there are statistically
significant differences among all groups. Ethanol (group 3) increased
the liver weight and the combination of ethanol and ephedrine + caffeine
(group 1) further increased the liver weight (means ± SE,
n = 5).
|
|
The ratio was significantly increased when ethanol was fed with ephedrine
and caffeine (group 1 compared with groups 2-4)
(P < 0.001). The ratio was also increased in the ethanol-fed rats
compared with the rats pair-fed dextrose (P < 0.001)
(Fig. 3). The liver weight not
corrected for body weight shows essentially the same pattern
(Fig. 4).
Blood catecholamines were determined in all four groups
(Fig. 5, A and
B). The blood catecholamine levels were elevated by
ethanol + ephedrine + caffeine (group A), by ethanol alone (group
C), and by ephedrine + caffeine alone (group B). The blood
catecholamine levels were total catecholamines, which included epinephrine and
norepinephrine but not dopamine. The elevation of the catecholamines in the
ephedrine + caffeine-fed group (B) was less than the ethanol-fed
group (group C). The combination of ethanol and ephedrine +
caffeine-fed group (group A) had significantly higher catecholamines
than the ephedrine + caffeine or ethanol-fed group (groups B and
C). When the levels of groups B and C were added
(group E), and compared with the combination of ethanol and ephedrine
+ caffeine (group A), group A values were significantly
higher. This indicates a synergistic effect when both ethanol and ephedrine +
caffeine were fed together. Epinephrine constituted more than half of the
total catecholamines except for group A where norepinephrine
predominated (Fig.
5B)

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5. A: total blood catecholamines are shown. Note that the baseline
values did not vary among groups (a-d) and the pair-fed dextrose group (d, D).
There was a significant increase in the experimental groups (A, B, C) (means
± SE, n = 35). B: norepinephrine (%total
catecholamines) is shown for each group as seen in A. Norepinephrine
is <50% of the total catecholamines except for group A in which it
was >50% of the total (mean ± SE, n = 35). Note that
there was a significant increase in the experimental groups A and
B (a vs. A and b vs. B; P < 0.05).
|
|
Liver damage was assessed by measuring serum alanine amino transferase
(ALT) and examining the histopathology of the liver in all four groups at the
beginning and end of the experiment. There was a significant increase in the
ALT levels in rats fed ethanol with ephedrine and caffeine (group 1)
when compared with rats fed ethanol alone without treatment (group 3)
(P < 0.001). Rats fed ethanol without treatment and the pair-fed
controls (group 3 vs. group 4) were also significantly
different (P < 0.001) (Fig.
6).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6. Serum alanine amino transferase (ALT) levels in all 4 experimental groups
(means ± SE, n = 5). Note that ethanol plus treatment
(group 1) shows a significant increase in ALT levels at the end of
treatment compared with the starting levels and compared with groups
2-4. Ethanol fed without ephedrine + caffeine treatment also
increased ALT levels (group 3) compared with their pair-fed controls
(group 4).
|
|
The histopathology of livers in the four experimental groups differed
(Fig. 7). Ethanol fed without
treatment (group 3) showed steatohepatitis
(Fig. 7) compared with the
normal histology of the pair-fed control (group 4)
(Fig. 7A). One liver
in group 3 had one small focus of centrilobular necrosis. The liver
cords were narrowed by ephedrine and caffeine with and without ethanol feeding
(groups 1 and 2) (Fig. 7,
C and D). In contrast to the livers of rats fed
ethanol without treatment (group 3) the livers of rats fed ethanol
with ephedrine and caffeine (group 1) showed only minimal
microvesicular fat. Instead, four of the rats showed focal centrilobular
ischemic necrosis (Fig. 7,
EG). One rat in group 1 also
showed blood-filled cysts without endothelial lining characteristic of
peliosis hepatitis (Fig.
7H). One rat in group 1 also showed scars, which
presumably were a result of prior episodes of ischemic necrosis
(Fig. 8,
AF).

View larger version (108K):
[in this window]
[in a new window]
|
Fig. 7. A: liver from a group 4 pair-fed control rat showing
normal histology hematoxylin and eosin (H & E); magnification x30.
B: liver from a group 3 rat fed ethanol showing
steatohepatitis H & E; magnification x30. C: liver from a
group 2 pair-fed rat treated with ephedrine and caffeine H & E
stain; magnification x30. D: liver from a group 1 rat
fed ethanol with ephedrine and caffeine; magnification x30.
EG: three foci from rats fed ethanol and treated with
ephedrine and caffeine show centrilobular ischemic necrosis H & E stain;
magnification x60. H: rat fed ethanol, ephedrine, and caffeine.
Note the blood cysts typical of peliosis hepatitis H & E stain;
magnification x60.
|
|

View larger version (133K):
[in this window]
[in a new window]
|
Fig. 8. Data from a rat fed ethanol, ephedrine, and caffeine (group 1)
(AF). A: daily UAL. B: liver
showing acute centrilobular ischemic necrosis, H & E stain; magnification
x30. C: liver showing centrilobular bridging fibrosis, H&E
stain; magnification x30. D: liver showing centrilobular
bridging fibrosis, reticulin stain, magnification x6. E: liver
showing centrilobular bridging fibrosis, reticulin stain; magnification
x30. F: liver showing a different focus, centrilobular bridging
fibrosis, reticulin stain; magnification x30.
|
|
Thus the ephedrine and caffeine diet prevented ethanol-induced
steatohepatitis, but the livers in this group (group 1) showed
centrilobular ischemic necrosis. Otherwise, the five livers in this group
resembled the pair-fed ephedrine and caffeine control livers (group
2).
The UAL cycle was present when the UALs were measured over the last 15 days
of the 6-wk feeding period studied in group 3
(Fig. 9). When the ethanol dose
was increased from 13
g·kg-1·day-1 to 14
or 15 g·kg-1·day-1
all five rats developed ethanol overdose as defined by death due to high
ethanol levels (14 ± 0.316) (Fig.
9).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9. The UAL levels of all 5 rats fed ethanol (group 3) are plotted.
Note that all the rats manifested the UAL cycle when given the 13
g·kg-1·day-1 ethanol
dose. Cycles were not synchronized so that peaks and troughs occurred on
different days in different rats. However, when the dose of ethanol was
increased to 14
g·kg-1·day-1, or in
the case of one rat 15
g·kg-1·day-1, the
rats developed ethanol overdose as defined by death from high ethanol
levels.
|
|
In contrast, in the rats fed ethanol, ephedrine, and caffeine (group
1), the UALs remained at a constant level between 200 and 300 mg/dl and
they did not cycle (Fig.
10).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 10. The UAL levels of all 5 rats fed ethanol, ephedrine, and caffeine
(group 1) are plotted. Note that the UAL cycle was completely
prevented by this treatment. When the dose of ethanol was increased from 13
g·kg-1·day-1 to 14
and then 15
g·kg-1·day-1, the
rats survived. Not until the ethanol dose of 16
g·kg-1·day-1 was
given did the rats develop ethanol overdose. One rat required a dose of 17
g·kg-1·day-1 before
eventually developing ethanol overdose.
|
|
When the dose of ethanol was increased from 13
g·kg-1·day-1, the
UALs did not begin to rise until 15
g·kg-1·day-1 was
given (Fig. 10). Ethanol
overdose did not develop until a dose of 1617
g·kg-1·day-1 of
ethanol was given (16.4 ± 0.245)
(Fig. 10). The difference
between the lethal dose of ethanol for groups 1 and 3 was
P < 0.001. Thus the ephedrine and caffeine treatment blocked the
UAL cycle and increased the elimination rate of ethanol so that the rats could
survive an otherwise lethal dose of ethanol. An example of the UALs of a rat
fed ethanol (group 3) and a pair-fed rat fed ethanol, ephedrine, and
caffeine is shown in Fig. 11.
This is shown so that the individual pair-fed rats can be compared with each
other.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 11. The UAL levels of one rat fed ethanol and its pair-fed rat fed ethanol,
ephedrine, and caffeine are plotted to better compare the data of individual
rats. Note that the rat fed ethanol (group 3) showed the UAL cycle at
a dose of ethanol at 13 g/day but overdosed when given 14
g·kg-1·day-1,
whereas, the rat fed ethanol, ephedrine, and caffeine did not develop the UAL
cycle and required a dose of 17
g·kg-1·day-1 ethanol
before overdosing.
|
|
 |
DISCUSSION
|
---|
As anticipated, ephedrine and caffeine feeding with ethanol prevented the
UAL cycle and increased the dose of ethanol, which the rats could tolerate.
The proposed explanation is that the increased metabolism caused by the
elevated blood catecholamine levels prevented the cyclic changes in the
metabolic rate caused by ethanol alone. This was similar to that seen when a
high dose of thyroxine was fed with ethanol
(7). That is, the
catecholamine-induced increase in metabolic rate accelerated the rate of
ethanol elimination, which prevented the urinary ethanol from reaching high
levels. When high levels were reached by increasing the dose of ethanol, the
hypothalamic-pituitary-thyroid mechanism of increasing the metabolic rate to
increase the ethanol elimination rate was desensitized by the already
increased metabolic rate. Thus the cycle was prevented from occurring.
Similar also to thyroxine feeding with ethanol, centrilobular ischemic
necrosis of the liver was caused by ethanol plus ephedrine and caffeine
feeding. This would suggest that the increased metabolic rate induced by
feeding thyroxine or ephedrine plus caffeine increased the hypoxia observed in
the liver at high blood ethanol levels as previously observed in the UAL cycle
when the intragastric ethanol feeding model was used
(1). The hypothesis that the
ischemic necrosis observed was due to hypoxia is on the basis of 1)
the fact that hypoxia of the liver has been documented at high blood ethanol
levels in this intragastric feeding rate model; and 2) the liver
morphology of the central necrosis resembles that seen in ischemic necrosis
observed in shock.
This observation may help explain the pathogenesis of liver toxicity
observed in patients taking ephedrine and caffeine as a treatment for obesity
(5). This phenomenon may put
patients at risk who drink alcohol and take adrenergic drugs for obesity.
Experimentally, an acute dose of ethanol causes an elevation of both
epinephrine and norepinephrine in mice
(6). There is, again, an
increase in plasma catecholamines in mice during ethanol withdrawal from
chronic ethanol feeding (6).
Thus in binge drinking the liver may be vulnerable to damage from hypoxia due
to elevated endogenous plasma catecholamines during acute ethanol ingestion
when high blood ethanol levels are achieved. This hypothesis is testable. In
the present report, blood catecholamines were increased more with ethanol
alone (group 3) compared with ephedrine plus caffeine fed alone
(group 2). The combination of ethanol and ephedrine plus caffeine
feeding (group 1) increased blood catecholamine levels indicating a
synergistic effect. This was associated with increased liver pathology due to
an enhanced degree of liver hypoxia that resulted.
 |
DISCLOSURES
|
---|
This study was supported by National Institute on Alcohol Abuse and
Alcoholism Grant AA-8116 and the Alcohol Center Grant on Liver and Pancreas
and Morphology Core of Keck Medical School of University of Southern
California, Los Angeles, CA.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Adriana Flores for typing the manuscript.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: S. W. French, Dept. of
Pathology, Harbor-University of California Los Angeles Medical Center, 1000 W.
Carson St., Torrance, CA 90509 (E-mail:
sfrench{at}rei.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
REFERENCES
|
---|
- Bardag-Gorce F,
French BA, Li J, Riley NE, Yuan QX, Reitz R, Cai Y, Wan Y-JY, and French
SW. The importance of cycling of blood alcohol levels in the pathogenesis
of experimental alcoholic liver disease fed ethanol intragastrically.
Gastroenterology 123:
325335, 2002.[ISI][Medline]
- Boozer CN,
Nasser JA, Heymsfield SB, Wang V, Chen G, and Solomon JL. An herbal
supplement containing Ma Huang-Guarana for weight loss: a randomized,
double-blind trial. Int J Obes
25: 316324,
2001.[ISI]
- Duloo AG and
Miller DS. The thermogenic properties of ephedrine/methylxanthine
mixtures: animal studies. Am J Clin Nutr
43: 388394,
1986.[Abstract]
- Dulloo AG and
Miller DS. Reversal of obesity in the genetically obese fa/fa Zucker rat
with an ephedrine/methylxanthines thermogenic mixture. J
Nutr 117:
383389, 1987.[ISI][Medline]
- Favreau JT, Ryu
ML, Braunstein G, Orshansky G, Park SS, Coody GL, Love LA, and Fong TL.
Severe hepatoxicity associated with the dietary supplement.
Lipokinetix Ann Intern Med 136:
590595, 2002.[Abstract/Free Full Text]
- Kovacs GL,
Soroncz M, and Tegyei I. Plasma catecholamines in ethanol tolerance and
withdrawal in mice. Eur J Pharmacol
448: 151156,
2002.[ISI][Medline]
- Li J, French
BA, Fu P, and French SW. Thyroid hormone causes liver necrosis in rats fed
ethanol intragastrically. Exp Mol Pathol
71: 7988,
2001.[ISI][Medline]
- Li J, Nguyen V,
French BA, Parlow AF, Su GL, Fu P, Yuan QX, and French SW. Mechanism of
the cyclic pattern of urinary ethanol levels in rats fed ethanol. The role of
the hypothalamic pituitary-thyroid axis. Am J Physiol Gastrointest
Liver Physiol 279:
G118G125, 2000.[Abstract/Free Full Text]
- Schola R and
Schwabe U. Stimulation of Ethanol Metabolism by Catecholamines
in Alcohol and Aldehyde Metabolizing Systems-IV, edited by
Thurman RG. New York: Plenum, 1980, p.
601618.
- Tsukamoto H,
French SW, Reidelberger RD, and Largman C. Cyclic pattern of blood alcohol
levels during continuous intragastric ethanol infusion in rats.
Alcohol Clin Exp Res 9:
3137, 1985.[ISI][Medline]
Copyright © 2003 by the American Physiological Society.