Metabolic Profile of Rats after One Hour of Intoxication with a Single Oral Dose of Ethanol
Manal A Hamed
The objective of this study was to mointer the metabolic disturbance in rats one hour after intoxication with one oral dose of ethanol. Rats orally administered with 10 g ethanol/kg body weight on 24 h empty stomach and sacrificed one hour later. Intoxicated rats recorded significant increase in blood alcohol level (alcohol dehydrogenase; ADH), aspartate (AST) and alanine (ALT) aminotransferase, lactate dehydrogenase (LDH) and gamma glutamyl transferase (GGT) enzyme activities. Carbohydrate deficient transferrin (CDT) level as a new marker for detection early independent ethanol intoxication recorded significant increase. Serum transferrin content was evaluated to avoid the false positive results of CDT. In conclusion, AST, ALT, LDH and GGT may be used as biomarkers for early diagnosis of ethanol toxicity. CDT plays a promise role for early diagnosis. CDT and the other biochemical tests especially GGT are recommended to be detected in parallel to improve the performance of either marker used alone.
Received: August 09, 2010;
Accepted: September 29, 2010;
Published: November 13, 2010
Alcohol is not digested like other foods. It avoids the normal digestive process
and goes directly to the blood stream (Levitt and Levitt,
1994). About 20% of the alcohol is absorbed directly into the blood through
the stomach walls and 80% is absorbed into the bloodstream through the small
intestine (Levitt et al., 1997). The maximum
absorption rate is obtained with the consumption of alcohol solution on an empty
stomach which also causes stomach ulceration (Karumi et
al., 2008). The absorption rate may be less when alcohol is consumed
with food (Horowitz et al., 1989).
Alcohol consumption leads to the production of the highly reactive ethanol
metabolite, acetaldehyde, which may affect intestinal tight junctions, increase
paracellular permeability and increase the solubility of penetrating chemicals
(Fisher et al., 2010). This enhancement is beneficial
if the chemicals involved are being used for therapeutic purposes, however exposure
to potentially toxic chemicals which occurs both in the workplace and at home
make increased absorption less desirable (Brand et al.,
The clinical evaluation in the early phase of alcohol misuse is rarely useful
for the diagnosis, since clinical signs are rather minimal (Sillanaukee,
1996). Furthermore, the common laboratory markers of alcohol misuse, such
as serum gamma glutamyltransferase (GGT), aspartate aminotransferase (AST),
alanine aminotransferase (ALT) and the erythrocyte Mean Cell Volume (MCV), have
been used as a diagnostic tool (Sharpe et al., 1996)
but with limited accuracy. Carbohydrate deficient transferrin, has high sensitivity
in detecting persons with alcohol dependence and shows promise for identification
of non-dependent hazardous drinking (0-70 g ethanol) (Schellenberg
et al., 2005).
The aim of the present study was to evaluate the changes of some biochemical parameters 1 h after intoxication with one oral ethanol dose (10 g kg-1 b.wt.) that may used as biomarkers for detecting toxicity and for early diagnosis.
MATERIALS AND METHODS
Animals handling: Male Wistar albino rats (100: 120 g) were selected for this study. They were obtained from the Animal House, National Research Center, Egypt. All animals were kept in a control environment of air and temp with access of water and diet. Animals left free for a week for acclimatization. All animals were deprived of food for 24 h before the beginning of the experiment with excess of water. All procedures followed the ethical guidelines of the Ethical Committee of the Federal Legislation and National Institutes of Health Guidelines in USA approved by the Medical Ethical Committee of the National Research Centre in Egypt.
Ethanol intoxication: Ten grams ethanol/kg body weight (Brand
et al., 2006) was orally given on 24 h empty stomach.
Experimental design: Thirty rats were divided into two groups. The first group (10 rats) served as healthy control group and orally administrated with 0.9 N physiological saline solution. The second group (20 rats) received the ethanol dose and sacrificed one hour after administration.
Serum sample: Blood collected from each animal by puncture the sub-tongual
vein in clean and dry test tube, left 10 min to clot and centrifuged at 3000
rpm for serum separation. The separated serum was stored at -80°C for further
Biochemical assays: Blood alcohol level was determined by the biochemical
assay of alcohol dehydrogenase (ADH) (Vallee and Hoch, 1955).
ADH catalyzes the oxidation of alcohol to acetaldehyde with the simultaneous
reduction of nicotinamide adenine dinucleotide (NAD) to NADH. The consequent
increase in absorbance at 340 nm is directly proportional to alcohol concentration
in the sample.
Aspartate and alanine amintransferases were measured by the method of Gella
et al. (1985), where the transfer of the amino group from aspartate
or alanine, formed oxalacetate or pyruvate, respectively. The developed colour
was measured at 520 nm.
Lactate dehydrogenase was assayed by the method of Babson
and Babson (1973), where the reduction of NAD is coupled with the reduction
of tetrazolium salt [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium
chloride] (INT). The resulted formazan of INT was measured colorimetrically
at 503 nm.
Gamma-Glutamyl Transferase (GGT): The assay measures the cleavage of
a specific GGT substrate (γ-glutamyl-p-ntiroanilide) by the enzyme to p-nitroaniline
(pNA) product which is proportional to the level of GGT enzyme in the sample.
The enzyme was measured at 405 nm (Szasz, 1969).
Serum CDT determinations were carried out using Kabi-Pharmacia kit (Sweden)
by the method of Stibler et al. (1991). This
test employs separation of transferrin isoforms on an anion exchange chromatography
microcolumn followed by quantification with double antibody radioimmunoassay.
Transferrin level was estimated by the method of Matsumoto
et al. (1991), where sandwich enzyme immunoassay of rat transferrin
with two monoclonal antibodies was applied. Microtiter plates coated with one
monoclonal antibody (15C2H3) were used and the captured transferrin was estimated
with a horseradish peroxidase-conjugated Fab' fragment of another monoclonal
Statistical analysis: All data were expressed as mean±SD of rat
numbers in each group. Statistical analysis was carried out using independent
student t-test (Ronald et al., 1983).
Work performance: The present study was done on March-April, 2010 at Therapeutic Chemistry Department, National Research Center, Cairo, Egypt.
RESULTS AND DISCUSSION
In the present study, blood alcohol concentration (200 ±8.16 ng mL-1)
in intoxicated rats recorded elevation by 53.84% as compared to the normal healthy
rats (Fig. 1). This was in accordance with Brand
et al. (2006) who observed a significant increase in blood alcohol
level two hour after one oral dose of ethanol. Blood alcohol concentration was
determined by measuring alcohol dehydrogenase activity, where in healthy rats
the normal substrates are not the alcohols or aldehydes that react most readily
with the enzyme but special substances such as vitamin A2 or farnesol (an intermediate
in cholesterol biosynthesis) (Krebs and Perkins, 1970). Obviously, the toxic
metabolic effects of ethanol oxidation are mainly due to increased liberation
of ROS, production of deleterious active acetaldehyde, increased NADH/NAD ratio
and disturbed intracellular calcium stores (Lieber, 2000;
Soliman et al., 2006). Therefore, ethanol forms
a toxic environment favorable to oxidative stress such as hypoxia, endotoxeamia
and cytokines release (Bautisa and Spitzer, 1999).
||Effect of ethanol intoxication on blood alcohol concentration.
Data are mean±SD of ten normal and twenty intoxicated rats. Blood
alcohol value is expressed as mg dL-1. Significant value at p≤0.05;
independent student t-test
||Effect of ethanol intoxication on serum AST, ALT and LDH.
Data are mean± SD of ten normal and twenty intoxicated rats. Enzyme
values are expressed as U L-1. Significant value at p≤0.05;
independent student t-test
Ethanol intoxicated rats recorded significant increase in AST (76.17±3.36
U L-1), ALT (33.96±3.12 U L-1) and LDH (133.29±3.66
U L-1) by 19.89, 22.24 and 16.03%, respectively (Fig.
2). In agreement with the present study Chen et al.
(2003) observed a significant increase in AST and ALT after light/moderate
drinkers (at least once per month; <210 g ethanol/week for men, <140 g
ethanol/week for women). In addition, Onyesom and Anosike
(2007) recorded elevation in AST and ALT in rabbits orally given 1.5 g ethanol
kg-1 body weight as single daily dose for a continuous period of
fifteen weeks. Moreover, Soliman et al. (2006)
recorded a significant decrease in rat liver LDH after chronic ethanol toxicity.
Although, AST, ALT and LDH are specific enzymes for detecting intoxication or
monitoring liver diseases, they lack the specificity for alcohol toxicity (Chen
et al., 2003). The increase in enzyme activities was mainly due to
the effect of ethanol that interpolates and expands biomembranes leading to
increase membrane fluidity and enzyme release (Yang et
al., 2005; Soliman et al., 2006).
CDT is the newest procedure available to clinicians to assess harmful alcohol
consumption and the first one to obtain approval from the US Food and Drug Administration
(FDA) for identification of sustained alcohol consumption. In contrast, GGT,
which is also FDA-approved, is intended to detect liver damage rather than alcohol
misuse (Harasymiw and Bean, 2001). CDT (17.83±2.46
U L-1) and GGT (32.10±2.46 U L-1) recorded enhancement
by 14.14 and 18.66%, respectively after intoxication with one oral dose of ethanol
(Fig. 3). Present results were confirmed by many workers who
postulated the use of CDT as a marker for heavily ethanol consumption (Harasymiw
and Bean, 2001; Chen et al., 2003; Koch
et al., 2004) neglecting to some extent its possible use as biomarker
for independent ethanol toxicity and for early diagnosis. Van
Pelt et al. (2000) documented the use of CDT and other biomarkers;
AST, ALT and GGT in parallel to improve the performance of either marker used
alone. They added that combination of CDT and GGT tests were more accuracy in
detecting toxicity. In addition, Van Pelt and Azimi (1998)
and Van Pelt et al. (2000) found that the amount
of CDT will also be influenced by the rate of transferrin synthesis.
Transferrin is a plasma protein that carries iron through the bloodstream to
the bone marrow, where red blood cells are manufactured, as well as to the liver
and spleen. Structurally, transferrin is a polypeptide with two N-linked polysaccharide
chains. These polysaccharide chains are branched with sialic acid (monosaccharide
carbohydrate) residues (Sharpe, 2001). Various forms
of transferrin exist, with differing levels of sialylation. The most common
form is tetrasialotransferrin, with four sialic acid chains.
||Effect of ethanol intoxication on serum GGT and CDT. Data
of GGT are mean±SD of ten normal and twenty intoxicated rats. CDT
values are mean±SD of eight normal and seventeen intoxicated rats.
Enzyme values are expressed as U L-1. Significant value at p≤0.05;
independent student t-test
||Effect of ethanol intoxication on serum transferrin level.
Data are mean±SD of ten normal and twenty intoxicated rats. Values
are expressed as ng mL-1. Significant value at p≤0.05; independent
In persons who consume alcohol, the proportion of transferrin with zero, one,
or two sialic acid chains is increased. These are referred to as carbohydrate-deficient
transferrin (Sharpe, 2001).
In the present study, transferrin level (170.00 ± 8.11 ng mL-1)
in intoxicated rats recorded significant increase by 13.33% (Fig.
4). This was in accordance with Golka et al.
(2004) who observed elevation in ferritin and transferrin levels after ethanol
intoxication. Van Pelt and Azimi (1998) and Bergstrom
and Helander (2008) postulated a significant correlation between CDT and
transferrin. Low iron status or high iron demand involves higher transferrin
synthesis, probably with a proportional increase of CDT isoforms. However, CDT
can induce false-positive outcome if the transferrin concentrations are low
as in anemic state. In the present work, rats with low transferrin level <5
ng mL-1 (Matsumoto et al., 1991) were
excluded to avoid false positive results of CDT. Control group under investigation
represented 33.30% of the total animals. Rats with transferrin level <5 ng
mL-1 were 6.66%. Intoxicated group represented 56.69% of the total
animals and 10.01% of them had low transferrin level (<5 ng mL-1)
(Fig. 5). Bergstrom and Helander (2008)
recorded some other factors influenced CDT level as end stage liver disease,
estrogens, pregnancy and inflammations. Wolff et al.
(2010) added that hemodialysis patients showed high CDT that seems to be
related to the low transferrin concentration associated with chronic kidney
||Percentages of animals with low and normal transferrin level.
Rats with normal transferrin value were subjected to CDT test
Present study was not concerned with these factors except transferrin level
as we used healthy animals. All animals showed no signs of liver or kidney diseases
or inflammations. Male animals let other factors like estrogen level and pregnancy
are impossibly to be concerned.
One oral dose of ethanol intoxication leads to a disturbance of certain metabolic parameters that can be used as markers for early detection of toxicity. Combination of tests especially GGT and CDT is recommended for accuracy and specificity. CDT can be used as a marker for independent hazadours ethanol toxicity and is suitable for early diagnosis of toxicity. Certain diseases must be taken into considerations to be sure of CDT accuracy.
Babson, A.L. and S.R. Babson, 1973.
Kinetic colorimetric measurement of serum lactate dehydrogenase activity. Clin. Chem., 19: 766-769.Direct Link |
Bautisa, A.P. and J. Spitzer, 1999.
Role of Kupffer cells in the ethanol induced oxidative stress in the liver. Frontiers Biosci., 4: 589-595.
Brand, R.M., J.L. Jendrzejewski, E.M. Henery and A.R. Charron, 2006.
A single oral dose of ethanol can alter transdermal absorption of topically applied chemicals in rats. Toxicol. Sci., 92: 349-355.CrossRef |
Chen, J., K.M. Conigrave, P. Macaskill, J.B. Whitfield and L. Irwig, 2003.
Combining carbohydrate-deficient transferrin and gamma-glutamyltransferase to increase diagnostic accuracy for problem drinking. Alcohol. Alcoholism., 38: 574-582.Direct Link |
Fisher, S.J., P.W. Swaan and N.D. Eddington, 2010.
The ethanol metabolite acetaldehyde increases paracellular drug permeability in vitro
and oral bioavailability in vivo
. J. Pharmacol. Exp. Ther., 332: 326-333.Direct Link |
Gella, F.J., T. Olivella, P.M. Cruz, J. Arenas, R. Moreno, R. Durban and J.A. Gomez, 1985.
A simple procedure for routine determination of aspartate aminotransferase and alanine aminotransferase with pyridoxal phosphate. Clin. Chem. Acta, 153: 241-247.PubMed |
Harasymiw, J. and P. Bean, 2001.
The combined use of the Early Detection of Alcohol Consumption (EDAC) test and carbohydrate-deficient transferrin to identify heavy drinking behaviour in males. Alcohol. Alcoholism, 36: 349-353.PubMed |
Horowitz, M., A. Maddox, M. Bochner, J. Wishart, R. Bratasiuk, P. Collins and D. Shearman, 1989.
Relationships between gastric emptying of solid and caloric liquid meals and alcohol absorption. Am. J. Physiol. Gastrointest Liver Physiol., 257: G291-G298.Direct Link |
Krebs, H.A. and J.R. Perkins, 1970.
The physiological role of liver alcohol dehydrogenase. Biochem. J., 118: 635-644.Direct Link |
Lieber, C.S., 2000.
Hepatic metabolic and nutritional disorders of alcoholism: From pathogenesis to therapy. Critisism Rev. Clin. Lab. Sci., 37: 551-584.Direct Link |
Levitt, M.D. and D.G. Levitt, 1994.
The critical role of the rate of ethanol absorption in the interpretation of studies purporting to demonstrate gastric metabolism of ethanol. J. Pharmacol., 269: 297-304.Direct Link |
Levitt, M.D., R. Li, E.G. Demaster, M. Elson, J. Furne and D.G. Levitt, 1997.
Use of measurements of ethanol absorption from stomach and intestine to assess human ethanol metabolism. Am. J. Physiol. Gastrointest. Liver Physiol., 273: G951-G957.Direct Link |
Matsumoto, T., H. Shima, T. Tetsuya Kishi and T. Sato, 1991.
Sandwich enzyme immunoassay for rat transferrin with two monoclonal antibodies and its application. Biochem. Med. Metab. Biol., 45: 188-196.CrossRef |
Ronald, T., S. Chapman and L. Hall, 1983.
Statistics in Research Development. The Chaucer Press Ltd., Bungay, Suffolk, New York and London, pp: 264-300
Schellenberg, F., R. Schwan, L. Mennetrey, M.N. Loiseaux, J.C. Pages and M. Reynaud, 2005.
Dose-effect relation between daily ethanol intake in the range 0-70 grams and % cdt value: Validation of a cut-off value. Alcohol. Alcoholism, 40: 531-534.CrossRef |
Sharpe, P.C., 2001.
Biochemical detection and monitoring of alcohol abuse and abstinence. Ann. Clin. Biochem., 38: 652-664.Direct Link |
Sharpe, P.C., R.D. McBride and G.P.R. Archbold, 1996.
Biochemical markers of alcohol abuse. Q. J. Med., 89: 137-144.
Stibler, H., S. Borg and M. Joustra, 1991.
A modified method for the assay of carbohydrate-deficient transferrin in serum. Alcohol. Alcoholism, 26: 451-454.
Szasz, G., 1969.
A kinetic photometric method for serum γ-glutamyl transpeptidase. Clin. Chem., 15: 124-136.PubMed |
Vallee, B. and F. Hoch, 1955.
Yeast alcohol dehydrogenase, a zinc metalloenzyme. J. Am. Chem. Soc., 77: 821-822.CrossRef |
Van Pelt, J. and H. Azimi, 1998.
False-positive CDTect values in patients with low ferritin values. Clin. Chem., 44: 2219-2220.Direct Link |
Van Pelt, J., G.L. Leusink, P.W.M. van Nierop and J.J. Keyzer, 2000.
Test characteristics of carbohydrate-deficient transferrin and -glutamyltransferase in alcohol-using perimenopausal women. Alcoholism Clin. Exp. Res., 24: 176-179.PubMed |
Yang, S.S., C.C. Huang, J.R. Chen, C.L. Chiu, M.J. Shieh, S.J. Lin and S.C. Yang, 2005.
Effect of antioxidant capacity in isolated rat hepatocytes. World J. Gastroenterol., 11: 7272-7276.
Wolff, F., M. Mesquita, F. Corazza, A. Demulder and D. Willems, 2010.
False positive carbohydrate-deficient transferrin results in chronic hemodialysis patients related to the analytical methodology. Clin. Biochem., 43: 1148-1151.PubMed |
Soliman, K.M., M.A. Hamed and S.A. Ali, 2006.
Hepatoprotective effect of carnosine on liver biochemical parameters in chronic ethanol intoxicated rat. J. Medical Sci., 6: 528-536.CrossRef | Direct Link |
Sillanaukee, P., 1996.
Laboratory markers of alcohol abuse. Alcohol Alcoholism, 31: 613-616.PubMed |
Onyesom, I. and E.O. Anosike, 2007.
Changes in Rabbit Liver function markers after chronic exposure to ethanol. Asian J. Biochem., 2: 337-342.CrossRef | Direct Link |
Koch, H., G. Meerkerk, J.O.M. Zaat, M.F. Ham, R.J. Scholten and W.J. Assendelft, 2004.
Accuracy of carbohydrate-deficient transferrin in the detection of excessive alcohol consumption: A systematic review. Alcohol Alcoholism, 39: 75-85.PubMed |
Karumi, Y., A.I. Augustine and I.A. Umar, 2008.
Gastroprotective effects of aqueous extract of Adansonia digitata
leaf on ethanol-induced ulceration in rats. J. Boil. Sci., 8: 225-228.CrossRef | Direct Link |
Golka, K., R. Sondermann, S.E. Reich and A. Wiese, 2004.
Carbohydrate-Deficient Transferrin (CDT) as a biomarker in persons suspected of alcohol abuse. Toxicol. Lett., 151: 235-241.PubMed |
Bergstrom, J.P. and A. Helander, 2008.
HPLC evaluation of clinical and pharmacological factors reported to cause false-positive Carbohydrate-Deficient Transferrin (CDT) levels. Clin. Chem. Acta, 389: 164-166.PubMed |