Division of Gastroenterology, Department of Internal Medicine and
1 Department of Orthopaedics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
(Received 4 March 2003; first review notified 2 April 2003; in revised form 4 May 2003; accepted 19 May 2003)
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ABSTRACT |
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INTRODUCTION |
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The first step in the metabolism of alcohol takes place in the hepatocytes, where conversion to acetaldehyde occurs. Acetaldehyde is then metabolised to acetate by aldehyde dehydrogenase (ALDH) (Bosron and Li, 1986; Bosron et al., 1993
; Lieber, 1994
). The toxic substance, acetaldehyde, and its derivatives are implicated in alcoholic cirrhosis of the liver (Goedde and Agarwal, 1989
). The liver alcohol dehydrogenase (ADH), ALDH, and cytochrome P4502E1 (P4502E1) are polymorphic at the ADH2, ADH3, ALDH2 loci, and the 5-flanking region of the P4502E1. Further, ethnic variations have been reported (Harada et al., 1980
; Smith, 1986
; Enomoto et al., 1991
; Thomasson et al., 1991
; Yoshida et al., 1991
; Goedde et al., 1992
; Kato et al., 1993
; Stephens et al., 1994
; Chao et al., 1995
). The ß2ß2 enzyme encoded by ADH2 2*2 is approximately 20-fold more active in ethanol oxidation than the ß1ß1 enzyme (Goedde et al., 1992
). Individuals who inherit the ADH2*2 allele have homodimeric and heterodimeric ß2-containing isozymes and could be expected to have faster rates of alcohol metabolism and possibly higher concentrations of acetaldehyde production after alcohol consumption. Many studies have attempted to explain susceptibility to alcoholism and alcohol induced liver disease in terms of differences in these alcohol-metabolizing enzymes (Harada et al., 1980
; Bosron and Li, 1986
; Enomoto et al., 1991
; Thomasson et al., 1991
; Goedde et al., 1992
; Bosron et al., 1993
; Chao et al., 1994a
; Maezawa et al., 1994
; Tsutsumi et al., 1994
; Yamauchi et al., 1995
). However, the specific mechanisms involved in the different alcohol-induced organ-specific diseases, such as those involving the liver, pancreas, heart and skeletal system, are not yet clear. Maezawa et al.(1996)
reported that Japanese alcoholic patients with more severe brain atrophy had a lower incidence of liver cirrhosis. Furthermore, that the ADH2*1 allele frequencies were different for the alcohol induced brain atrophy and cirrhosis subpopulations. These results suggest that subpopulations of alcoholic patients defined by organ specific complications are probably genetically different. In our previous study, it was also demonstrated that Chinese patients with alcohol-induced cirrhosis and acute pancreatitis constituted two genetically distinct subpopulations, with many alcohol induced cirrhosis patients never experiencing acute alcoholic pancreatitis despite the fact that the daily alcohol intake of the former was significantly higher than that of the latter (Chao et al., 1997
). Further analysis revealed that the ADH2*1 allele frequency for the pancreatitis patients was significantly lower than for their cirrhotic counterparts. Differences in ADH2*1 and/or ALDH2*1 allele frequencies have also been demonstrated for Chinese alcoholic patients diagnosed with oesophageal cancer in comparison to analogues with acute pancreatitis and cirrhosis (Chao et al., 2000
).
Avascular necrosis of the hip joint (AVN) is a specific complication of chronic alcoholism. Clinically, we have noted that this complication was more common for alcoholic patients with liver disease than for those with acute pancreatitis. In this study of alcoholic patients, therefore, we wanted to establish whether the allele frequencies for the alcohol metabolizing enzyme genes for the AVN subgroup are different from those in other alcoholic subgroups and whether they were more similar to the liver cirrhosis subgroup than alcoholic-pancreatitis analogues.
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PATIENTS AND METHODS |
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ADH2, ADH3 and ALDH2 genotyping and detection of the RsaI and PstI polymorphisms of the P4502E1 gene
DNA was extracted from white blood-cell pellets, obtained after lysis of the red cells with ammonium bicarbonate. The ADH2 and ADH3 genotypes were determined accord-ing to the method of Groppi et al. (1990), with minor modifications (Chao et al., 1994a
,b
). Briefly, the primers 247 (5'GAAGGGGGGTCACCAGGTTG) and HE45 (5'AATCTTTTCTGAATCTGAACAG) were used for amplification of the ADH2 genes, and the primers 321 (5'GCTTTAAGAGTAAATATTCTGT CCCC) and YC351 (5'AATCTACCTCTTTCCAGAGC) were used for amplification of the ADH3 genes. The PCR-amplification programme for both the ADH2 and ADH3 is set at 35 cycles, with each cycle consisting of 1 min at 95°C, 45 s at 55°C, and 45 s at 72°C.
ALDH2 genotyping was determined by our previously published method, using PCR-directed mutagenesis (Chao et al., 1994a, 1997
). The RsaI and PstI polymorphisms of the P4502E1 gene were determined according to the method of Hayashi et al.(1991)
.
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STATISTICAL ANALYSIS |
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RESULTS |
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As 16 of the alcoholic AVN subgroup had either been diagnosed with alcoholic liver disease or a history of alcoholic pancreatitis, only the ADH2 and ALDH2 genotypes and allele frequencies for the remaining 35 patients (those with just one complication, alcoholic AVN-1) were used in the comparison with the other alcoholic subgroups (Tables 3,4
). Alcoholic patients with both AVN and pancreatitis had more similar ADH2 and ALDH2 allele frequencies than did their counterparts with cirrhosis. The ADH2*1 and ALDH2*2 frequencies were similar for the alcoholic cirrhosis patients and their counterparts with both AVN and cirrhosis.
For further evaluation whether the differences in allele frequency are biologically important, the distribution of ADH2 genotypes in the different alcoholic groups have been compared (Table 3). The results were similar to the allele frequency comparisons.
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DISCUSSION |
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Despite the fact that alcoholic AVN patients had a higher incidence of coexistent alcoholic liver disease, surprisingly the ADH2 and ALDH2 allele frequencies for the alcoholic AVN and acute pancreatitis subgroups were more similar than for the alcoholic AVN and cirrhosis subgroups. The allele frequency of ADH2*1 was only significantly different between patients with alcoholic AVN and cirrhosis of the liver and was not significantly different between patients with alcoholic AVN and pancreatitis. Further analysis showed that the ADH2*1 and ALDH2*2 frequencies were similar for the alcoholic cirrhosis patients and the counterparts with both AVN and cirrhosis (two complications) (Table 4). Analysis of these data suggests two possibilities. First, alcoholic patients with similar ADH2*1 and ALDH2*1 genetic patterns may acquire AVN or pancreatitis. Therefore, in addition to the alcohol-metabolizing enzyme genes we studied, other genetic variations may also influence the type of organ complication in alcoholic patients. An ever-accumulating body of clinical and experimental evidence indicates that inflammatory responses are involved in the pathogenesis of alcoholic liver disease. The association between alcoholic liver disease and polymorphism of the CD14 endotoxin receptor gene, tumour necrosis factor promotor polymorphism and interleukin 10 promotor region polymorphism has been reported (Grove et al., 1997
, 2000
; Jarvelainen et al., 2001
). Second, alcoholic patients with both AVN and cirrhosis (two complications) may be another specific subpopulation that is different from alcoholic patients with AVN only. Alcoholic patients with higher ADH2*1 allele frequency are prone to both AVN and cirrhosis and patients with lower ADH2*1 allele frequency are likely to have AVN only.
By comparing the allele frequencies for the alcohol-metabolizing enzyme genes, we have confirmed that alcoholic patients may be stratified into genetically different subpopulations defined by organ-specific diseases. Previously, we reported that alcoholic patients with acute pancreatitis and oesophageal carcinoma are different from their counterparts with cirrhosis, in terms of ADH2*1 and ALDH2*2 allele frequency. In the present study, we have shown that Chinese alcoholic patients with AVN are different from cirrhotic and oesophageal carcinoma subgroups in terms of ADH2*1 and/or ALDH2*2 allele frequencies. Interestingly, it was also shown that, although the mean age of alcoholic patients with AVN was greater and the daily alcohol consumption significantly higher than for those with pancreatitis, most of the former had never had pancreatitis. These findings suggest that they constituted a distinct subpopulation. Indeed, although statistical significance was not achieved, the ADH2*1 allele frequency was lower for the alcoholic AVN than for the alcoholic pancreatitis subgroup. In fact, the alcoholic AVN subgroup had the lowest ADH2*1 allele frequency of the alcoholic subpopulations studied.
Phenotypic differences between homozygous ADH2 2*2 and ADH2 1*1 is that the former alcoholics have faster ethanol metabolism and consequently may have faster acetaldehyde accumulation, which also depends on the ALDH activity. However, according to the present data, we do not consider acetaldehyde accumulation plays the only major role determining the formation of AVN.
It is well known that tobacco use is also a confounding variable in development of the alcohol-induced organ damage mentioned in this context. In this study, more than 90% of the alcoholics were also smokers. Hence, we cannot clearly answer the question of how the smoking status might impact on our results. In a clinical study it is difficult to control all variables such as drinking pattern, age of onset of alcoholism, sex and coincidence of alcoholic pathologies, which are important in exploring the relationship between polymorphism of ADH and ALDH genes and specific alcohol-induced organ damage.
Although it is now possible to genotype the alcohol-metabolizing enzyme genes and to determine the specific gene polymorphisms responsible for particular inflammatory processes (Grove et al., 1997, 2000
; Jarvelainen et al., 2001
), the probability of individual complications still cannot be estimated. It seems highly probable that the development of specific complications in alcoholic patients is determined by multiple genes, with most of these still not well understood.
In conclusion, allele frequencies of ADH2*1 and ALDH2*2 appear to differ among disease-defined subpopulations of Chinese alcoholics, suggesting that differences in the alcohol-metabolizing enzyme genes account, at least in part, for the different organ-specific complications. Clinically, alcoholic AVN coexisted more frequently with alcoholic liver disease than with acute alcoholic pancreatitis. However, comparing ADH2 and ALDH2-allele frequencies for the alcohol-induced disease subgroups, the AVN and acute pancreatitis patients were more similar than their AVN and cirrhosis counterparts, suggesting that other genetic polymorphisms may also influence the type of organ-specific complications in chronic alcoholic patients.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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