Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid)

Clinica Chimica Acta 356 (2005) 35 – 57 www.elsevier.com/locate/clinchim

Review

Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid) Tetsuya YamamotoT, Yuji Moriwaki, Sumio Takahashi Division of Endocrinology and Metabolism, Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1. Nishinomiya, Hyogo 663-8501, Japan Received 13 December 2004; received in revised form 29 January 2005; accepted 31 January 2005

Abstract There are many factors that contribute to hyperuricemia, including obesity, insulin resistance, alcohol consumption, diuretic use, hypertension, renal insufficiency, genetic makeup, etc. Of these, alcohol (ethanol) is the most important. Ethanol enhances adenine nucleotide degradation and increases lactic acid level in blood, leading to hyperuricemia. In beer, purines also contribute to an increase in plasma uric acid. Although rare, dehydration and ketoacidosis (due to ethanol ingestion) are associated with the ethanol-induced increase in serum uric acid levels. Ethanol also increases the plasma concentrations and urinary excretion of hypoxanthine and xanthine via the acceleration of adenine nucleotide degradation and a possible weak inhibition of xanthine dehydrogenase activity. Since many factors such as the ALDH2*1 gene and ADH2*2 gene, daily drinking habits, exercise, and dehydration enhance the increase in plasma concentration of uric acid induced by ethanol, it is important to pay attention to these factors, as well as ingested ethanol volume, type of alcoholic beverage, and the administration of anti-hyperuricemic agents, to prevent and treat ethanol-induced hyperuricemia. D 2005 Elsevier B.V. All rights reserved. Keywords: Uric acid; Hypoxanthine; Xanthine; Purine bases; Ethanol; Alcohol

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . 2. Epidemiological background . . . . . . . . . . . 3. Mechanism of ethanol-induced increases in levels 3.1. Adenine nucleotide degradation . . . . . . 3.1.1. Overproduction of acetyl-CoA . .

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Abbreviations: ADH, alcohol dehydrogenase; MEOS, microsomal ethanol-oxidizing system; ALDH, aldehyde dehydrogenase; Pi, inorganic phosphate; URAT1, urate transporter 1; ALDH2, mitochondrial aldehyde dehydrogenase. T Corresponding author. Fax: +81 798 45 6472. E-mail address: [email protected] (T. Yamamoto). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2005.01.024

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T. Yamamoto et al. / Clinica Chimica Acta 356 (2005) 35–57

3.1.2. Reduction in redox potentials of nicotinamide adenine nucleotides . . . . . . . . . . . . . 3.1.3. NADH-induced inhibition of xanthine dehydrogenase activity . . . . . . . . . . . . . . . 3.2. Increased lactic acid in blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Dehydration and ketosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Purines present in alcoholic beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Consumption of lead-contaminated alcoholic beverages . . . . . . . . . . . . . . . . . . . . . . . 4. Relationships of aldehyde dehydrogenase (ALDH)2 gene and alcohol dehydrogenase (ADH)2 gene with ethanol-induced increase in plasma concentration of oxypurines . . . . . . . . . . . . . . . . . . . . . . 5. Relationship between plasma ethanol and uric acid levels . . . . . . . . . . . . . . . . . . . . . . . . . 6. Factors other than genetic that have an effect on the ethanol-induced increase in plasma purine bases. . . 6.1. Daily drinking habits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Dehydration (decreased extracellular volume) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Drugs (pyrazinamide, furosemide) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1. Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.2. Ingestion of purine-rich foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Prevention and treatment of ethanol-induced hyperuricemia . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Restriction of ingested ethanol volume. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Restriction from purine-containing alcohol beverages . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Change in drinking habits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Weight control, restriction of purine-rich and high calorie food, restriction of rigorous exercise, and for dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Administration of anti-hyperuricemic agents (allopurinol and benzbromarone) . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction While heavy alcohol consumption by gouty patients has been noted in many studies [1,2], the association between heavy alcohol consumption and increased risk of gout has been suggested by epidemiological findings [3–7]. A direct relationship between increased serum uric acid levels and alcohol consumption was demonstrated by Lieber [8]. Lieber administered alcohol to non-gouty heavy drinkers and found an increase in blood lactic acid and uric acid levels, along with a concomitant decrease in urinary uric acid excretion. Based on these findings, Lieber proposed that lactic acid formed during metabolism of alcohol interfered with the urinary excretion of uric acid, thus leading to increased plasma concentration. Thereafter, ethanol-enhanced abrupt ATP consumption was also proposed as a mechanism involved in the rise in levels of plasma purine bases (hypoxanthine, xanthine, and uric acid) [9,10]. During the course of metabolism of alcohol, ATP is abruptly

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39 42 43 45 46 47

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consumed, followed by its degradation to uric acid (ATPYADPYAMPYIMPYinosineYhypoxanthineYxanthineYuric acid) (Fig. 1). Several other substances, such as fructose and xylitol, as well as exercise also abruptly consume ATP and cause an increase in plasma purine base levels [11–17]. These facts strongly suggest that a rise in serum uric acid levels due to ethanol is due to abrupt ATP consumption. In addition, since alcohol beverages contain the various concentrations of purine content that affect serum uric acid levels [18], it has been suggested that the purine content in alcohol beverages might also contribute to the hyperuricemic effect of ethanol and an increase in the risk of gout, though definite data has only become available recently. Over the past 30 years, studies have shown that the ethanol-induced rise in serum uric acid levels is related to not only ingested ethanol volume, but also to the type of alcoholic beverage, daily drinking habits, and genetic makeup such as the ALDH2 gene [19–21]. In addition, beer ingestion following rigor-

T. Yamamoto et al. / Clinica Chimica Acta 356 (2005) 35–57

ATP

Purine nucleotide de novo

ADP S-AMP AMP Adenosine Adenine

37

Nucleic acid

XMP GMP

IMP

Guanosine

Inosine Hypoxanthine

Xanthine

Guanine

Uric acid Fig. 1. Map of adenine nucleotide degradation. Bold arrows show the adenine nucleotide degradation pathway. IMP=inosine 5V-monophosphate, GMP=guanosine 5V-monophosphate.

ous exercise raises serum uric acid levels synergistically, as the amount was reported to be greater than the sum of increases by beer ingestion and exercise alone [22]. On the basis of these findings, life style considerations must also be made to prevent the condition of ethanol-induced hyperuricemia which causes gout. In this review, we focus on current epidemiological, basic, and clinical evidence linking the effects of ethanol on purine bases, as well as the enhancing factors for ethanol-induced hyperuricemia, and prevention and treatment for ethanolinduced hyperuricemia.

2. Epidemiological background According to a US National Health Interview Survey (NHIS) conducted on 1996 [23], the overall prevalence of gout is 4.6% in men and 2% in women, for a ratio of approximately 2:1 of men to women. Results from 1983 to 1985, however, indicated that the self-reported prevalence rate for all ages was 1.36% in men and 0.64% in women [24]. The incidence of gout is known to parallel increasing uric acid levels and [25]. In subjects with a serum uric acid concentration greater than 9.0 mg/dL, the 5-year cumulative incidence was nearly 25% [25]. These data strongly suggest that hyperuricemia is the most important cause of gout. Hyperuricemia is thought to be ascribable to genetic makeup as well as other factors. Thus far, gene mutations responsible for inherited diseases that cause hyperuricemia, such as hypoxanthine-guanine

phosphoribosyl transferase deficiency, 5-phosphoribosyl-1-pyrophosphate synthetase over-activity, aldolase-beta deficiency, glucose-6-phosphatse deficiency, and abnormal uromodulin, have been reported [26– 28]. It is likely, however, that many other mutations responsible for hyperuricemia remain to be identified. Other factors that contribute to hyperuricemia include high body mass index (BMI), visceral fat obesity, insulin resistance, hypertension, alcohol consumption, diuretic use, and renal insufficiency [4–6,24–43]. Of these, alcohol consumption is probably the most well known. Previously, Garrod stated that the use of fermented liquors is the most powerful of all predisposing causes of gout [2]. Further, Drum et al. proposed that hyperuricemia is a marker for ethanol ingestion [44]. Other studies noted that alcohol consumption was increased in patients with gout compared to controls [45,46]. In contrast, prospective studies reported that alcohol consumption was not significantly associated with gout [4,6,7]. These studies, however, were limited by a small sample size and lack of comprehensive adjustment of relevant variables. Recently, Choi et al. demonstrated that alcohol consumption was significantly associated with uric acid level [47]. This prospective study included 47,150 men with no history of gout showed that alcohol consumption was strongly associated with an increased risk of gout (Table 1) [47]. In addition, they found that the risk of gout varied depending on type of alcoholic beverage. For example, beer increased the risk of gout more than twice as much as did spirits, even though the alcohol content was less in beer than spirits (12.8 g vs. 14.0 g). Interestingly, two 4-oz glasses of wine or more per day was found not to be

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Table 1 Relative risk for gout incidence according to daily alcohol intake Alcohol intake

Number of participants Person-years Age-adjusted relative risk Multivariate relative riska

P for trend

0

0.1–4.0

5.0–9.9

10.0–14.9

15.0–29–9

30.0–49.9

N50

105 94691 1.0 (reference) 1.0 (reference)

164 131093 1.13 (0.89–1.45) 1.09 (0.85–1.40)

109 75844 1.30 (0.99–1.70) 1.25 (0.95–1.64)

90 58643 1.37 (1.03–1.81) 1.32 (0.99–1.75)

126 69090 1.63 (1.26–2.11) 1.49 (1.14–1.94)

97 37331 2.30 (1.75–3.04) 1.96 (1.48–2.60)

39 11241 3.02 (2.09–4.36) 2.53 (1.73–3.70)

b0.001 b0.0001

Values in parentheses are 95% CI. a Adjusted for age, total energy intake, body mass index, diuretic use, history of hypertension, history of renal failure, intake of total meats, sea food, purine-rich vegetables, dairy foods, and fluids.

associated with risk of gout. These findings suggest that the large purine content present in beer may play an important contributory role in the incidence of gout. Despite these findings, other non-alcoholic risk factors in beer and protective factors in wine remain to be characterized.

3. Mechanism of ethanol-induced increases in levels of plasma purine bases Ethanol ingestion increases serum uric acid levels by increasing the production of uric acid and decreasing its renal clearance. By increasing the production of oxypurines, ethanol also increases plasma hypoxanthine and xanthine levels and urinary excretion [9,10]. However, the ethanol-induced rise in plasma concentration of purine bases is suggested to occur via several mechanisms, which are described below.

suggested that adenine nucleotide degradation occurs during these metabolic processes [9,10,50–52]. 3.1.1. Overproduction of acetyl-CoA Faller and Fox showed that daily uric acid turnover during long-term oral ethanol intake (1.8 g/kg/day) increased from a baseline value of 10 mg/dL to 170% of the baseline value in patients with gout [9]. In addition, the serum level of uric acid increased from a mean baseline of 8.4 to 10.1 mg/dL, with an increase in the ratio of uric acid clearance to creatinine clearance of 145% The ratio of urinary oxypurines to creatinine also increased to 641% of the baseline value. The results obtained in this study indicated that uric acid production was increased by ethanol ingestion, thus leading to an increase in the level of uric acid. However, it remains unclear whether uric acid production due to ethanol is increased by an elevation of de novo purine synthesis or by the acceleration of purine nucleotide degradation. In order

3.1. Adenine nucleotide degradation Oral ethanol intake leads to absorption by diffusion in the small intestine. Following absorption, ethanol becomes distributed in total body water and subsequently metabolized by the liver. Ethanol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH), the microsomal ethanol-oxidizing system (MEOS), and catalase, and is subsequently oxidized to acetate by aldehyde dehydrogenase (ALDH) [48] (Fig. 2). Acetate formed from ethanol in the liver is mostly transported to the peripheral tissues and then metabolized to CO2 and H2O [49]. Many studies have

(ADH) NAD NADH Ethanol

Acetaldehyde

(MEOS) NADPH

(Mitochondrial ALDH) NAD NADH Acetate

(Cytosol ALDH)

NADP

Extrahepatic oxidation Hepatic oxidation

NAD NADH (Catalase) HO

HO

Fig. 2. Ethanol elimination pathway. Bold arrows show the main pathway.

T. Yamamoto et al. / Clinica Chimica Acta 356 (2005) 35–57

to address this question, they performed a 2-h intravenous ethanol infusion study (0.25 to 0.35 g/kg/h) after isotopic adenine administration and found that urinary radioactivity increased to 129% and 149% of the baseline value during successive hours of ethanol administration. Furthermore, the ratio of urinary uric acid to creatinine increased to 114% and 123%, while that of urine oxypurines to creatinine increased to 341% and 415% [9]. Those results strongly suggested that the production of uric acid and its precursors is increased by accelerated degradation of adenine nucleotides. However, a contribution to increased uric acid synthesis by de novo purine synthesis could not be ruled out. Indeed, a secondary activation of de novo purine synthesis would be expected to replace the process of adenosine triphosphate being converted to purine end products because ethanol increases phosphoribosyl pyrophosphate, a rate-limiting substrate for de novo purine synthesis [53]. Therefore, to determine the mechanism of ethanol-induced degradation of adenine nucleotides, Puig and Fox infused ethanol in a 5–10% solution at the rate of 100 ml/h and acetate in an 8–10% solution at the rate of 100 ml/ h (0.1 mmol/kg/min) into normal subjects following isotopic adenine administration [10]. Infusion of sodium acetate at a rate of 0.1 mmol/kg/min for 1 h increased plasma acetate from a mean baseline value of 0.04 mM to 0.35 mM. Equimolar ethanol infusion also increased from a mean baseline value of 0.04 mM to 0.08 mM, while the oxypurines-to-creatinine ratio increased to peak values of 223% of the baseline value and 316% of the baseline value after acetate and ethanol infusions, respectively. In addition, urinary radioactivity increased to 171% and 128% of the baseline value after acetate and ethanol infusions, respectively. In contrast, the ratio of urinary uric acid to creatinine and that of uric acid clearance to creatinine clearance were not decreased after ethanol and acetate infusions. Together, these results indicate that both acetate and ethanol increase the excretion of the uric acid precursor and labeled degradation products derived from the adenine nucleotide pool. Ethanol is oxidized to acetate via acetaldehyde in the liver and then is mostly transported to peripheral tissues [49] (Fig. 2). Accordingly, it is possible that acetate formed from ethanol may play a role in ethanol-induced adenine nucleotide degradation in peripheral tissues and the liver. Peak plasma acetate

39

levels following acetate infusion were approximately 4-fold higher as those following ethanol infusion. The exact quantitative responses of urinary oxypurines and urinary radioactivity after acetate and ethanol administration were, however, different in the study of Puig and Fox [10]. Acetate is mainly metabolized to acetyl-CoA via acetyl-AMP. During this conversion (acetateYacetylAMPYacetyl-CoA), ATP is dephosphorylated to AMP. Because ATP is converted to AMP in this pathway, 2 mol of high-energy phosphate are consumed for each mole of ethanol metabolized. Although most of the AMP formed is resynthesized to ATP, a small amount of AMP may enter the pathway of adenine nucleotide degradation (AMPY IMPYinosineYhypoxanthineYxanthineYuric acid), thus leading to uric acid production (Fig. 3). This proposed mechanism is very attractive and has been frequently cited in many articles related to ethanolinduced hyperuricemia. 3.1.2. Reduction in redox potentials of nicotinamide adenine nucleotides As described above, ethanol is oxidized to acetaldehyde mainly by ADH in the cytoplasm of liver cells, coupled with a reduction of NAD to NADH, and partly by MEOS in the microsomes of liver cells, coupled with an oxidation of NADPH to NADP. Subsequent oxidation to acetate (mainly by ALDH in the mitochondria and partly by ALDH in the cytoplasm) is coupled to reduction of NAD to

Ethanol

NAD

Uric acid

NADH Acetaldehyde

NAD

Xanthine

NADH Acetate

Hypoxanthine

ATP PPi

Inosine

Acetyl AMP IMP

CoASH AMP Acetyl CoA

Fig. 3. Proposed mechanism for adenine nucleotide degradation during ethanol metabolism. PPi=inorganic pyrophosphate, Pi=inorganic phosphate, IMP=inosine 5V-monophosphate.

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NADH (Fig. 2). During these oxidative processes, the redox potentials of NAD, both in the cytoplasm and mitochondria of hepatocytes, are reduced. The interference of liver metabolism by ethanol is to a large extent due to the NADH + H+ produced in these processes. In the mitochondria, the electron transfer system allows the reoxidation of NADH + H+ to NAD+. In the cytosol, reoxidation of NADH involves malate/aspartate and irreversible dihydroxyacetone phosphate/sn-glycerol-3-phosphate shuttles because the mitochondrial membrane is impermeable to NADH. In addition, reoxidation of cytosolic NADH, without the transfer of reducing equivalents to the mitochondria, is, in part, due to reduction of pyruvic acid to lactic acid. Nevertheless, ethanol increases the ratio of lactic/pyruvic acid in blood [52,54–56], which reflects the increased ratio of NADH/NAD in the liver cytosol. This increased ratio also indicates a reduction in the redox potentials of NAD in cytosol. In a perfused rat liver study that utilized NMR spectroscopy [57], glycolysis was inhibited during ethanol-induced reduction in the redox potentials of NAD in cytosol (Fig. 4), as evidenced by a decrease in lactic + pyruvic acid release, increased snglycerol-3-phosphate, and decreased Pi. Consequently, the total hepatic content of nucleoside 5Vtriphosphates, which indicated a catabolism of

Lactic acid TCA Acetyl CoA NAD NADH Pyruvic acid ATP Acetic acid 3 p-glycerate ADP NADH 1, 3 bp-glycerate AMP NAD IMP Acetaldehyde NADH HxR Glyceraldehyde-3-p NAD Hx Ethanol DHAP X NADH Glucose NAD UA (Liver) Glycerol sn 3-phosphate Fig. 4. Proposed mechanism for enhanced adenine nucleotide degradation by the reduction in redox potentials of nicotinamide adenine nucleotides in the cytosol of liver cells. Glycolysis is inhibited together with sn-glycerol-3-phosphate accumulation, resulting in an enhancement of adenine nucleotide degradation. DHAP=dihydroxyacetone phosphate, IMP=inosine 5V-monophosphate, HxR=inosine, Hx=hypoxanthine, X=xanthine, UA=uric acid.

adenine nucleotides, was decreased (Table 2) [57]. In another perfused rat liver study [58], glycolytic ATP production was also diminished [58]. Those results strongly suggest that ATP production in mitochondria in the presence of ethanol is insufficient to alleviate the inhibition of glycolytic ATP production in cytosol, though mitochondrial ATP synthesis was demonstrated to increase in ethanol-perfused whole livers [59]. Abrupt ATP consumption reflects enhanced adenine nucleotide catabolism (ATPYADPYAMPYIMPY inosineYhypoxanthineYxanthineYuric acid) (Fig. 1). The rate-limiting enzyme of the adenine nucleotide catabolism pathway is AMP deaminase, which is inhibited by inorganic phosphate [60]. Ethanol increases sn-glycerol-3-phosphate, which traps inorganic phosphate (Pi) during a reduction in the redox potentials of NAD in cytosol (Fig. 4). A decrease in the concentration of inorganic phosphate in the cytosol relieves the inhibition by inorganic phosphate toward AMP deaminase leading to accelerated adenine nucleotide catabolism. In previous in vitro studies [61], acetaldehyde, a metabolite of ethanol, increased hypoxanthine level in erythrocyte incubation medium. Decreased ATP and increased AMP, ADP, and glyceraldehyde 3phosphate + dihydroxyacetone phosphate was found in erythrocytes that did not possess mitochondria, but did contain acetaldehyde dehydrogenase (Table 3). However, in erythrocytes lacking aldehyde dehydrogenase activity, acetaldehyde did not have any effect on glycolysis or adenine nucleotide production (Table 3). There was also no affect on normal erythrocytes by acetate [61]. In addition, another study [52] found that the addition of pyruvate to erythrocyte incubation medium prevented the acetaldehyde-induced changes described above, indicating that the added pyruvate was converted to lactic acid by lactate dehydrogenase together with the conversion of NADH to NAD in erythrocytes. These results suggest that the conversion of NAD to NADH plays an important role in glycolysis and glycolytic ATP production (Fig. 5). Because acetaldehyde is metabolized to acetate, coupled with the conversion of NAD to NADH in erythrocytes, it is strongly suggested that an inhibition of the reduction in redox potentials of NAD by pyruvate prevents acetaldehyde-induced

T. Yamamoto et al. / Clinica Chimica Acta 356 (2005) 35–57

41

Table 2 Metabolic rates and NTP depletion in perfused rat livers following ethanol administrationa Ethanol (mM) 70 (n = 8) (1) (2) (3)

0.35 F 0.05 0.31 F 0.04 0.96 F 0.03

10 (n = 16) 0.34 F 0.03 0.30 F 0.06 0.95 F 0.09

2.0 (n = 10) 0.26 F 0.05 0.23 F 0.07 0.75 F 0.07

1.0 (n = 9) 0.17 F 0.04 0.13 F 0.06 0.46 F 0.06

0.5 (n = 8)

0 (n = 3)

0.11 F 0.04 0.10 F 0.06 0.23 F 0.05

b b

0.18 F 0.04

(1) initial rate of G3P accumulation [Amol min 1 (g wet of liver) ], (2) initial rate of Pi depletion [Amol min 1 (g wet of liver) ], (3) NTP depletion (NMR) [Amol min 1 (g wet of liver) ]. The control value of NTP was 2.88 F 0.02 Amol (g wet wt of liver) 1 (n = 51). G3P and NTP denote sn-glycerol-3-phosphate accumulation and nucleoside triphosphates, respectively. NTP depletion nearly matched ATP loss [50]. a The initial rates of sn-glycerol 3-phosphate accumulation and Pi depletion were calculated from 31 P NMR data obtained 2 min following ethanol administration. NTP depletion was determined by the difference between the control level (2.88 F 0.02 Amol/g of liver wet wt) and NTP levels at the end of the experiment, which was 20 min after ethanol administration. Data are expressed as mean F S.E.M. b Not measurable.

inhibition of glycolysis, glycolytic ATP production, and adenine nucleotide degradation. Actually, following ethanol exposure, the concentrations of AMP, ADP, and glyceraldehyde 3-phosphate + dihydroxyacetone phosphate in erythrocytes increased and that of pyruvic acid in blood decreased significantly, with a concomitant increase in the plasma concentration and urinary excretion of oxypurines in healthy subjects [52]. These findings suggested that acetaldehyde-induced adenine nucleotide degradation in erythrocytes may contribute to the increase in plasma concentration of oxypurines by ethanol, though the effect seemed to be slight. Based on the results described above, it is suggested that in the liver cytosol, as in erythrocytes, a decrease in the

concentration of NAD during the oxidation of ethanol to acetate via acetaldehyde inhibits glycolysis at the step of glyceraldehyde-3-phosphate conversion to 1, 3-bisphosphoglycerate, and accumulates both glyceraldehydes-3-phosphate and dihydroxyacetone phosphate, while an increase in the cytosol concentration of NADH accelerates the conversion of dihydroxyacetone phosphate to snglycerol-3-phosphate. This mechanism effectively traps inorganic phosphate (Pi) thus leading to decreased inorganic phosphate. Therefore, it is also considered that inhibition of glycolysis reduces glycolytic ATP synthesis and a decrease in the concentration of inorganic phosphate in the cytosol enhances adenine nucleotide degradation (Fig. 4).

Table 3 In vitro effect of acetaldehyde on adenine nucleotides and glyceraldehydes-3-phosphate + dihydroxyacetone phosphate (GA3P + DHAP) (nmol/ ml erythrocytes) in erythrocytes and hypoxanthine (mg/ml) in incubation medium Acetaldehyde Control Aldehy dedehydrogenase-deficient erythrocytes Hypoxanthine 0.14 AMP 17.4 ADP 112 ATP 1200 GA3P + DHAP 14

2 Ag/ml

4 Ag/ml

40 Ag/ml

0.14 19.4 110 1256 14

0.15 18.2 113 1231 15

0.14 21.5 118 1220 15

0.19 F 0.07T 33.0 F 6.6T 153 F 15T 1291 F120T 59 F 24T

0.70 F 0.36T 160.0 F 64.8T 272 F 64T 861 F163T 230 F 38T

Normal erythrocytes possessing aldehyde dehydrogenase activity (mean F S.D., N = 20) Hypoxanthine 0.10 F 0.05 0.14 F 0.06T AMP 19.2 F 4.5 27.0 F 5.4T ADP 113 F 9 137 F 10T ATP 1390 F 123 1333 F 124T GA3P + DHAP 14 F 6 36 F 17T T P b 0.05 compared with the control.

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Acetaldehyde Glucose

NAD NADH

Glyceraldehyde-3-p NADH NAD Gro 3P Inosine

IMP

Hypoxanthine

Acetic acid

AMP

1, 3 bp-glycerate ADP (Erythrocytes) ATP 3 p-glycerate

ADP PEP ATP Lactic acid Pyr NAD

NADH

Fig. 5. Proposed mechanism for enhanced adenine nucleotide degradation during oxidation of acetaldehyde in erythrocytes. Glycolysis is inhibited, shown by dashed line, via NAD consumption accompanied by the conversion of acetaldehyde to acetic acid. Glycolysis is also inhibited by the reduction in redox potentials of nicotinamide adenine nucleotides due to the oxidation of acetaldehyde, resulting in an enhancement of adenine nucleotide degradation. Large bold arrows indicate adenine nucleotide degradation. Gro 3P=sn-glycerol-3-phosphate, PEP=phosphoenol pyruvate, Pyr=pyruvic acid. Bold arrows indicate the enhanced adenine nucleotide degradation pathway.

3.1.3. NADH-induced inhibition of xanthine dehydrogenase activity In many studies [9,10,19,22,52,54–56], ethanol has been shown to increase the plasma concentration and urinary excretion of oxypurines. An increase in the plasma concentration and urinary excretion of oxypurines, which is thought to be due to ethanolinduced adenine nucleotide degradation, such as that induced by exercise. During rigorous exercise, adenine nucleotide degradation occurs in muscle tissue that rarely possesses xanthine dehydrogenase. This limitation leads to a marked increase in the plasma concentration of hypoxanthine (the endproduct of adenine nucleotide degradation in muscle) and a small increase in the plasma concentration of xanthine [17,22,62]. Following ethanol ingestion, adenine nucleotides degradation appears to occur mainly in the liver, which does possess xanthine dehydrogenase. This leads to a considerable increase in the plasma concentrations of hypoxanthine and xanthine. Furthermore, the increases in plasma concentration and urinary excretion of xanthine are greater than those of hypoxanthine [10,20,22,52,54–

56], and the same as produced by administration of small amounts of allopurinol (xanthine oxidase inhibitor). In a previous study [63], ethanol decreased the conversion of allopurinol to oxypurinol in humans by an unknown mechanism (Fig. 6). Allopurinol is a xanthine dehydrogenase (xanthine oxidase) inhibitor, and is itself oxidized to oxypurinol by xanthine dehydrogenase and aldehyde oxidase [64–67]. These results suggest that ethanol may inhibit xanthine dehydrogenase activity, though only slightly. To examine the effect of ethanol on xanthine dehydrogenase activity, pyrazinamide was administered together with ethanol in our previous study [54]. Although pyrazinamide (an antituberculosis drug) is metabolized to pyrazinoic acid by microsomal deamidase and subsequently to 5-hydroxypyrazinoic acid by xanthine dehydrogenase (classic pathway), metabolism can also occur via an alternate pathway to 5-hydroxypyrazinamide by xanthine dehydrogenase and aldehyde oxidase [54,68–72] (Fig. 7). In the subjects who received pyrazinamide, ethanol ingestion decreased the plasma concentration and urinary excretion of 5-hydroxypyrazinamide and 5-hydroxypyrazinoic acid (Fig. 8). Pyrazinamide and pyrazinoic acid were not changed. These results also strongly suggested that xanthine dehydrogenase activity was inhibited in relation to the metabolism of ethanol. Therefore, the ethanol-induced increases

(A)

(B)

Ethanol ingestion µmol/l

µmol/l

40

Ethanol ingestion

40 * *

20

*

20 *

0

6

12

24 (hr)

0

6

12

24 (hr)

Fig. 6. Effect of ethanol on metabolism of allopurinol (n = 3). (A) and (B) show the plasma concentrations of allopurinol and oxypurinol, respectively. The open circle shows the intake of allopurinol (200 mg) together with whiskey (200 ml), and the closed circle the intake of allopurinol (200 mg) alone. *P b 0.05.

T. Yamamoto et al. / Clinica Chimica Acta 356 (2005) 35–57

Microsomal deamidase Pyrazinoic aid

Pyrazinamide

Xanthine dehydogenase

Xanthine dehydogenase

Aldehyde oxidase 5-hydroxypyrazinoic aid

5-hydroxypyrazinamide

Fig. 7. Major pathways of pyrazinamide metabolism.

in plasma concentration and urinary excretion of oxypurines may be due to a combination of a modest ethanol-induced inhibition of xanthine dehydrogenase and increased adenine degradation, though ethanol-induced increases in the plasma concentration and urinary excretion of oxypurines have been regarded as evidence of increased purine degradation. On the other hand, the primary effect of ethanol on uric acid is thought to be an increase in the plasma concentration of uric acid, since ethanolinduced adenine nucleotide degradation and an

(A)

(B) Ethanol ingestion

Ethanol ingestion µmol/l

mmol/hour

20

0.4 *

*

*

*

0.2

10 *

-30

30

*

-1 90 (min)

1

increase in the blood concentration of lactic acid play a greater role than that of ethanol-induced inhibition of xanthine dehydrogenase. It is also important to elucidate the mechanism of inhibition of xanthine dehydrogenase activity. Several previous studies [73,74] have shown that NADH inhibits xanthine dehydrogenase activity (Fig. 9). NADH is produced by the reduction of NAD, which is coupled with the conversion of ethanol to acetaldehyde and successively that of acetaldehyde to acetate. During these reactions, the cytosolic concentration of NAD decreases, that of NADH increases, and, consequently, the ratio of NADH/NAD increases in liver tissue possessing xanthine dehydrogenase. Therefore, increased cytosolic NADH may directly inhibit the activity of xanthine dehydrogenase present in the cytosol (Fig. 9). In addition, decreased cytosolic NAD (a substrate of xanthine dehydrogenase) may inhibit the oxidation of oxypurines by xanthine dehydrogenase, especially that of xanthine to uric acid, because xanthine dehydrogenase has a higher Km value for xanthine than hypoxanthine. In fact, in subjects who received pyrazinamide, the plasma concentration and urinary excretion of 5-hydroxypyrazinamide and 5-hydroxypytrazinoic acid were decreased by xylitol infusion which increased the ratio of NADH/NAD, as was also seen following ethanol ingestion [75].

*

3.2. Increased lactic acid in blood *

0

43

2 (hr)

Fig. 8. Effects of ethanol ingestion on plasma concentration and urinary excretion of 5-hydroxypyrazinamide and 5-hydroxypyrazinoic acid (n = 6). Pyrazinamide (0.48 mmol/kg body weight) was administered 11 h before ethanol ingestion. (A) and (B) show the plasma concentrations and urinary excretion of 5-hydroxypyrazinamide and 5-hydroxypyrazinamide, respectively. The closed circle shows the plasma concentration and urinary excretion of 5hydroxypyrazinamide following ethanol ingestion, the closed square shows the plasma concentration and urinary excretion of 5-hydroxypyrazinamide with no ethanol ingestion, the open circle shows the plasma concentration and urinary excretion of 5hydroxypyrazinoic acid following ethanol ingestion, and the open square shows the plasma concentration and urinary excretion of 5hydroxypyrazinamide with no ethanol ingestion. * denotes P b 0.05, as compared with before ethanol ingestion.

Previously, Lieber demonstrated that ethanol ingestion increased the concentration of lactic acid in blood and that of uric acid in serum, and also decreased the urinary excretion of uric acid, suggesting that the increase in serum concentration of uric acid is due to the increase in the blood concentration of lactic acid [8] (Fig. 10). Lactic acid is an organic acid that is secreted via the organic acid transport system in the Ethanol

Acetaldehyde

NAD NADH Hypoxanthine

NAD

Xanthine

Acetic acid NADH Uric acid

Fig. 9. Proposed mechanism of inhibition of xanthine dehydrogenase activity by NADH. The dashed line denotes the inhibition of xanthine dehydrogenase activity.

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Alcohol ingestion 300 200 100 21 0 mg/dl 12 6 mg/dl

Ethanol level Lactate level

mg/dl

mg/min

9 8 7

Urate level

8 ml/min

Urate clearance

0.4 0.3 0.2 0.1

4 Urinary urate excretion 2

4

6

8

10

12

36

(hr)

Fig. 10. Relationship between lactic acid and uric acid.

kidneys. In a previous study using the racemic form of lactate [76], it was demonstrated that its infusion decreased uric acid clearance in humans. Thereafter, it was also demonstrated that the l-lactate decreased uric acid clearance [77]. Uric acid is freely filtered through the glomerulus and approximately 90–95% of filtered uric acid is reabsorbed [78]. Despite its resorption, uric acid is also actively secreted into the proximal tubular lumen [79,80]. Recent studies have suggested that the secretion of uric acid contributes minimally to excreted uric acid and that excreted uric acid largely represents filtered uric acid that has escaped the reabsorption [81]. In many studies [1,45,46,76], it has been suggested that lactic acid reduces the renal excretion of uric acid by competitively inhibiting its secretion by the proximal tubule. However, recently it was also suggested that lactic acid may accelerate the reabsorption of uric acid by the proximal tubule [82,83]. The results of membrane vesicle studies [82,84–93] have suggested that the uric acid exchanger (electroneutral uric acid/anion exchanger) and voltage-sensitive pathway (electrogenic uric acid uniporter) are located in both the apical and basolateral membranes of proximal tubule cells. However, no transporters have been found that regulate serum uric acid levels and urinary excretion. Recently, four proteins were identified at the molecular level that transport uric acid [83,93–95], one of which was cloned and termed as urate transporter 1 (URAT1 encoded by SLC22A12) by Enomoto et al. [83]. They demonstrated that URAT1 is located at the apical membrane in the proximal tubule and its defect caused renal hypouricemia,

indicating that URAT1 regulates serum uric acid. Furthermore, it was shown that lactic acid in the proximal tubular cells was transported into the proximal tubular lumen by URAT1 in coordination with the transport of uric acid in the proximal tubular lumen into proximal tubular cells in vitro. These findings strongly suggest that the excretion of lactic acid accelerates the reabsorption of uric acid in the proximal tubule via URAT1 (Fig. 11). Accordingly, it is possible that URAT1 is a main contributor of a lactic acid-induced decrease in the urinary excretion of uric acid, though other unidentified transporters may be involved. Excessive ethanol ingestion, rigorous exercise, and von Gierke’s disease (glucose-6-phosphatase deficiency) each increase the concentration of lactic acid in blood leading to an increased serum concentration of uric acid [8,16,17,96,97]. Although it is well known that exercise and glucose-6phosphatase deficiency enhance glycolysis, resulting in hyperlactatemia [96,97], it remains unclear why ethanol increases the blood concentration of lactic acid. Ethanol is oxidized to acetaldehyde and consequent oxidation to acetate. During this oxidation, NAD is reduced to NADH + H+ in the cytosol of liver cells. The increased ratio of NADH/NAD accelerates the reduction of pyruvic acid to lactic acid coupled with the oxidation of NADH + H+ to NAD. Therefore, it is suggested that an ethanolinduced increase in the ratio of NADH/NAD enhances lactic acid production in the liver. HowTubular lumen Proximal tubular cell

Na+

Anion

URAT1

Anion Lactate Nicotinate pyrazinoate

Urate

Brush border

Basolateral

[ Proposalmechanism] model ] [Proposed

Fig. 11. Proposed mechanism for urate transport via URAT1 at the brush border.

T. Yamamoto et al. / Clinica Chimica Acta 356 (2005) 35–57

45

found that it was decreased by approximately 0.37and 0.12-fold at blood lactic acid levels of 2.40 and 3.09 mM, respectively [77]. These results indicated that an ethanol-induced increase in blood concentration of lactic acid is a factor that contributes to the ethanol-induced increase in plasma concentration of uric acid. Besides the effect of lactic acid on uric acid, it is possible that xanthine is transported by URAT1, as many studies have suggested that uric acid and xanthine share the transport system in part [99–102]. However, it was also shown that l-lactate infusion did not affect the urinary excretion of xanthine and hypoxanthine [77]. Accordingly, other transporter(s) may be shared by uric acid and xanthine.

ever, as described previously in Section 3.1.2 (reduction of redox potentials of NAD), a perfused rat liver study demonstrated that lactic + pyruvic acid release from the liver was decreased by ethanol (Fig. 12), suggesting that lactate production was decreased by ethanol-induced glycolytic inhibition [57]. Under the condition of an ethanol-induced increase in the ratio of NADH/NAD, it is possible that the oxidation of lactic acid to pyruvic acid by lactate dehydrogenase diminishes in the liver, resulting in a decrease in the uptake of lactic acid in blood into the liver. In an in vivo study in cats [98], it was demonstrated that hepatic lactic acid uptake was suppressed with increased ethanol level in blood, suggesting that decreased lactic acid uptake in the liver may cause an ethanol-induced increase in the blood concentration of lactic acid. However, further examination is needed, since the mechanism of such an ethanolinduced increase in the blood concentration of lactic acid remains undetermined. In any case, ingestion of a large amount of ethanol raises the blood concentration of lactic acid, leading to a decreased urinary excretion of uric acid. In the ethanol ingestion study of Lieber et al. [8], the urinary excretion of uric acid diminished together with an increase in its blood concentration to 20 mg/ dL (2.22 mmol/L). Therefore, we examined the effect of lactic acid on the urinary excretion of uric acid and

3.3. Dehydration and ketosis If a sufficient amount of alcohol is ingested, diuresis occurs followed by dehydration. The most attractive explanation for ethanol-induced diuresis is that ethanol ingestion inhibits the release of antidiuretic hormone [103]. Previously, it was reported that diuresis and volume depletion induced by alcohol consumption resulted in decreased glomerular filtration and increased tubular reabsorption of uric acid, while fat-dependent ketosis that occurs with long-term ingestion might diminish the urinary excretion of uric acid [1].

(nmol x mini-1x g-1)

Lactate+Pyruvate release

1300

1100 0.5 mM

900

Ethanol

1 mM

700

2 mM 10/70 mM

500

300 -10

-6

-2

2

6

10

14

18

22

Time (min)

Fig. 12. Effect of ethanol concentration on lactic acid + pyruvic acid release. Effluent from perfused livers was sampled every 4 min for the release of lactic acid and pyruvic acid. After a 30-min equilibration period, the perfusion medium was supplemented with ethanol (arrow). Values for lactic acid + pyruvic acid release are presented as mean F S.E.M., for n experiments: n = 8 (70 mM ethanol), n = 16 (10 mM ethanol), n = 10 (2 mM ethanol), n = 9 (1 mM ethanol), n = 8 (0.5 mM ethanol).

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Heavy ingestion of alcoholic beverages may lead to the development of ketoacidosis. Dillon et al. reported that severe alcoholics had similar findings (heavy ketonuria and low serum CO2) as patients with ketoacidotic diabetes [104] and most of those alcoholics were chronic ethanol overusers, who sometimes become acutely starved because of a lack of food intake owing to gastric intolerance or to an intercurrent acute illness [105]. The precise mechanisms of alcoholic ketoacidosis, especially that of increased lipolysis, remain undetermined. However, several factors are known to promote ketogenesis in patients who show signs of starvation and have recently ingested ethanol [105]. Ketoacids are weak organic acids that presumably share the renal transport system with uric acid. Thus, it has been suggested that the tubular secretion of uric acid is inhibited by 3-hydroxybutyrate and acetoacetate [105]. In a recent in vitro study, it was shown that lactate and acetoacetate were secreted by URAT1, in combination with the reabsorption of uric acid [83]. Therefore, increased plasma ketoacids may enhance the reabsorption of uric acid by the proximal tubule, leading to a decreased urinary excretion of uric acid. However, hyperuricemia induced by dehydration or ketoacidosis due to ethanol ingestion seems to be rare, since the ingestion of alcoholic beverages increases the plasma concentration of uric acid without evidence of dehydration and ketoacidosis in most cases. 3.4. Purines present in alcoholic beverages The ingestion of beer or other types of alcoholic beverage is known to increase the plasma concentration of uric acid [56,106]. Although ethanol itself increases the plasma concentration of uric acid, purines present in alcohol beverages may increase the plasma concentration of purine bases (uric acid, hypoxanthine, xanthine). Beer contains a relatively high amount of purines as compared with other alcoholic beverages (Table 4) [18]. In a previous study [46], Gibson et al. found that patients with gout ingested greater amounts of alcoholic beverages than control subjects. Although the daily intake of most nutrients, including total purine nitrogen, was similar, 41% of the subjects with gout consumed more than 60 g of alcohol daily. Further, it was demonstrated

that the intake of purine nitrogen, half of which was derived from beer, was higher in those with gout who consumed more than 60 g of alcohol daily. From those results, it was suggested that the effect of ingested purine, half of which was derived from beer, had a clinical effect on serum uric acid, augmenting the hyperuricemic effect. In another study [18], Gibson et al. demonstrated that ingestion of beer containing 53 g of ethanol and 70.6 mg of purine nitrogen over 4 h increased the plasma concentration of uric acid, though an equal volume of artificial fruit juice sweetened with glucose did not, which suggested that the purines contained in beer along with ethanol cause increases in the levels of plasma and urinary uric acid. In addition, Gibson et al. suggested that the relatively large amounts of guanosine present in beer may be more quickly reabsorbed and rapidly converted to uric acid in humans, since guanosine is more readily absorbed than other nucleosides, nucleotides, or bases in animals [46]. In a recent study [56], regular beer (10 ml/kg body weight) increased the plasma concentration of uric acid by approximately 30 Amol/L, while freeze-dried beer (0.34 g/kg body weight) also increased that by approximately 20 Amol/L. Since 10 ml of regular beer contains 0.34 g of content when freeze-dried, the amount of purines in freeze-dried beer are the same as those in regular beer. These results strongly suggest that the purines in beer have an effect on plasma uric acid levels. In another study [19], it was demonstrated that beer (0.8 ml ethanol equivalent/kg body weight) increased the serum concentration of uric acid by 13.6%. On the other hand, whiskey or shouchu (Japanese distilled liquor) (0.8 ml ethanol equivalent/kg body weight), which contain very low levels of purines, did not affect the serum concentration of uric acid [19]. Recently, low malt liquor (happo-shu) and purine-free happo-shu have been developed in Japan. The former contains lower amounts of malt and a considerable amount of purines, while the latter contains the same lower amounts of malt and very low level of purines [106]. In our study of these two kinds of happo-shu, regular happo-shu (10 ml/kg body weight) increased the plasma concentration of uric acid and purine-free happo-shu did not, clearly indicating that the purines present in regular happo-shu caused an increase in the plasma

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Table 4 Average purine content of seven traditional British beers (expressed as mg purine nitrogen/l): results are compared with values for Home-brewed beer, Guinness, and Large beer Beverage

Adenine

Hypoxanthine

Xanthine

Adenosine

Guanosine

Total purines

British beer (range) Home-brewed beer Guinness Lager beer

1.2 (0.4–3.0) 0.7 0 4.3

0.6 (0–2.5) 1.2 0 3.0

3.3 (2.6–3.8) 0.2 5.5 3.5

3.5 (0–6.0) 0 7.7 0

13.6 (10–17.3) 1.8 10.6 6.9

22.2 (20.3–27.5) 3.9 23.8 17.7

concentration of uric acid. That study also demonstrated the effects of purines in regular happo-shu on the plasma concentration and urinary excretion of oxypurines (hypoxanthine and xanthine). The urinary excretion of hypoxanthine and xanthine, as well as that of uric acid, increased following ingestion of regular happo-shu. Further, the plasma concentration of xanthine following ingestion of regular happo-shu increased, as compared with purine-free happo-shu, though the plasma concentration of hypoxanthine with regular happo-shu was not significantly different from that following the ingestion of purine-free happo-shu. Those results indicated that purines in regular happo-shu increased the production of oxypurines. On the other hand, freeze-dried beer did not have an effect on the plasma concentrations and urinary excretion of hypoxanthine and xanthine, suggesting that the purines in freeze-dried beer were converted to uric acid via hypoxanthine and xanthine in the liver and small intestine in the absence of ethanol [56]. As described in Section 3.1.3, these results suggest that xanthine dehydrogenase activity is slightly inhibited during the metabolism of ethanol. 3.5. Consumption of lead-contaminated alcoholic beverages Clinical lead poisoning gives rise to recognized, but nonspecific syndromes [107–109], which include musculoskeletal (gout), renal (interstitial nephritis), and hematopoietic syndromes [109]. Ingestion of lead leads to small-sized kidneys, interstitial and periadvential fibrosis, and slowly progressive renal failure. In addition, lead poisoning reduces renal uric acid excretion, leading to hyperuricemia. Homemade alcoholic beverages were reported as a source of lead in the southeastern United States, as

automobile radiators, which have many lead-soldered connections, are often used, since they are readily available and inexpensive [109]. Further, to improve the taste of fermented wines in France, lead plates were sometimes added. Consumption of those types of alcoholic beverages may cause clinical lead poisoning, leading to hyperuricemia.

4. Relationships of aldehyde dehydrogenase (ALDH)2 gene and alcohol dehydrogenase (ADH)2 gene with ethanol-induced increase in plasma concentration of oxypurines ALDH2 genotypes are known to regulate the sensitivity of an individual to ethanol. The common allele ALDH2*1 codes for normal ALDH2 activity, while the less common allele ALDH2*2 codes for a lower ALDH2 activity and widely distributed in Mongoloid populations including Japanese, in contrast to Caucasoid populations [110–113]. Individuals homozygous for ALDH2*2 (ALDH2*2/ALDH2*2) rapidly respond to ethanol ingestion, and show such symptoms as facial flushing and palpitation, because they cannot oxidize acetaldehyde to acetate, resulting in the rapid accumulation of acetaldehyde [110]. Therefore, such individuals rarely drink alcoholic beverages because of the adverse effects. On the other hand, individuals homozygous for ALDH2*1 (ALDH2*1/ALDH2*1) are able to oxidize acetaldehyde to acetate because of their normal ALDH activity, leading to a rapid metabolism of ethanol. This effectively increases the ratio of NADH/NAD and accelerates adenine nucleotide degradation. A study found that ingestion of 60 ml of whiskey increased the plasma concentration of hypoxanthine to higher levels in subjects with ALDH2*1/ ALDH2*1 than in those with ALDH2*1/ALDH2*2, while the concentration was not increased in subjects

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with ALDH2*2/ALDH2*2 [21]. This finding suggests that the ALDH2 gene is a contributing factor in the ethanol-induced increase in plasma concentration of hypoxanthine. Furthermore, it was suggested that rapid ethanol metabolism is a key factor for purine metabolism (enhanced adenine nucleotide degradation and slightly inhibited xanthine dehydrogenase activity). Homozygotes with ALDH2*2 comprise approximately 10% of Japanese and other Asian populations [113]. In Japan, homozygotes with the ALDH2*1 genotype are more frequently found among gouty patients than normal subjects, while those with the ALDH2*2 genotype are less often found in gouty patient populations as compared to normal [21]. Therefore, Japanese gout patients may easily develop a habit of alcohol ingestion because they are generally able to consume large quantities without ill effects. In contrast, Hashimoto et al. showed that the frequency of individuals with a uric acid value in the highest group was significantly higher in those with ADH2*2/ADH2*2 genotypes than in those with the ADH2*1/ADH2*1 genotypes [114]. No difference was observed between the subjects with ALDH2*1/ ALDH2*1 genotypes and those with ALDH2*1/ ALDH2*2 or ALDH2*2/ALDH2*2 with respect to the frequency of individuals with serum uric acid in the highest group. ADH is an important enzyme that oxidizes ethanol to acetaldehyde and six ADH genes have been characterized. Among them, the ADH2 and ADH3 loci are polymorphic. The ADH2 gene polymorphism plays an important role in individual variations regarding ethanol elimination, as ADH2*1 encodes for the h1 subunit with low activity, ADH2*2 encodes for the h2 subunit with high activity, and ADH2*3, which is rarely detected in Japanese, encodes for the h3 subunit. Therefore, Hashimoto’s results suggest that the ALDH2 genotype is not related to an increase in serum concentration of uric acid due to ethanol ingestion. In contrast, the ADH2 genotype, which plays an important role in ethanol metabolism, appears to have such a relationship. Hyperuricemia is a polygenic disease and its phenotype is influenced by many factors. Although alcohol ingestion is one of those factors that substantially contributes to hyperuricemia, drinking habits are controlled by a variety of genes.

5. Relationship between plasma ethanol and uric acid levels The relationships between blood ethanol concentration and psychomotor activity have been studied and are well known. Despite these well-documented studies, the precise relationships between blood ethanol and serum uric acid concentrations remain unclear [8–10,18,19,52,56]. According to the results of those ethanol ingestion studies, the blood concentration of ethanol is largely correlated to an increase in the serum concentration of uric acid. When ethanol in blood is below 6.5 mmol/l (30 mg/dL), the serum concentration of uric acid is unaltered although urinary excretion of oxypurines is increased [9,10]. When blood concentration of ethanol is elevated from 6.5 to 10 mmol/L (30 to 50 mg/dL), the serum concentration of uric acid scarcely changes although urinary excretion of uric acid and oxypurines increases [19]. However, with the same ethanol concentration following the ingestion of beer (which contains a relatively large amount of purines), the plasma concentration of uric acid is increased by less than 30 Amol/L (0.5 mg/dL) [56]. When the blood concentration of ethanol ranges from 10 to 20 mmol/L (50 to 100 mg/dL), the serum concentration of uric acid does not change [9]. However, in the range of 20 to 45 mmol/L, serum uric acid is increased by 13% to Table 5 Relationship between blood ethanol concentration and purine bases Blood ethanol level Less than 30 mg/dl

Purine bases (oxypurines and uric acid) in blood and urine

no change in serum uric acid level, increase in urinary oxypurine excretion [10] 30 to 50 mg/dl no change in serum uric acid level, increase in urinary oxypurine excretion [19,52], increase in plasma uric acid level by beer [56], increase in urinary purine bases excretion [56] 50–100 mg/dl no change in serum uric acid level at 100 mg/dl [9] 100–200 mg/dl increase in serum uric acid level by 13 to 24% during beer ingestion [18], increase in urinary uric acid excretion by 7 to 20% during beer ingestion [18] More than 200 mg/dl increase in serum uric acid level by 20%, decrease in urinary uric acid excretion together with increase in blood concentration of lactate [8]

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24% following beer ingestion and urinary uric acid excretion is increased by 7% to 20% [18]. In addition, when the blood concentration of ethanol becomes greater than 45 mmol/L (200 mg/dL), the serum concentration of uric acid increases by about 20% [8], as summarized in Table 5.

6. Factors other than genetic that have an effect on the ethanol-induced increase in plasma purine bases 6.1. Daily drinking habits Habitual alcohol drinking accelerates ethanol metabolism and thereby may enhance ethanol-induced adenine nucleotide degradation. In a previous study [20], Nishimura et al. demonstrated that regular drinkers, who consumed more than 60 g of ethanol every day, consumption of 0.5 g of ethanol/kg increased serum uric acid levels by 52.6 Amol/L (0.8 mg/dL). This finding was not, however, seen in nondrinkers/occasional drinkers who consumed less than 20 g of ethanol. They also demonstrated that hypoxanthine and xanthine in both plasma and urine and serum acetate were increased more in regular drinkers than in nondrinkers/occasional drinkers. Those results may explain why rapid ethanol metabolism accelerates adenine nucleotide degradation via enhanced ethanol oxidation and slightly inhibits dehydrogenase activity, due to ethanol ingestion. Interestingly, these findings suggest that accelerated adenine nucleotide degradation secondary to enhanced ethanol oxidation may cause ethanol-induced hyperuricemia in regular drinkers. 6.2. Exercise Many patients with gout have experienced a gouty attack following muscular exercise, such as tennis or football, as well as after ethanol ingestion. Rigorous exercise accelerates adenine nucleotide degradation in exercising muscles together with lactic acid production, causing an increase in the plasma concentration of purine bases [16,17]. In a previous study [96], Yamanaka et al. showed that adenine nucleotide degradation and lactic acid production were enhanced when exercise was beyond the anaerobic threshold,

49

which indicated that the time of increased hypoxanthine production due to increased adenine nucleotide degradation in exercising muscles was consistent with that of lactic acid production. This finding suggested that both play an important role in the increase in plasma concentration of uric acid. In Japan, it is common for people to drink alcoholic beverages, especially beer, after exercise. Although that apparently increases the plasma concentration of purine bases to a greater degree than alcohol ingestion or exercise alone, the amount of increase remains to be precisely determined. We recently demonstrated that a combination of beer (10 ml/kg body weight) ingestion and exercise for 30 min at 70% of maximum oxygen uptake synergistically increased the plasma concentration of uric acid by 87 Amol/L (1.46 mg/dL), and the amount was greater than the sum (58 Amol/L, 0.97 mg/dL) of increases by beer ingestion and exercise alone [22]. In addition, it was demonstrated that the blood concentration of lactic acid that increased by exercise decreased slowly during ethanol metabolism in our combination experiment, as compared with the exercise alone experiment, while creatinine clearance decreased in both the combination and exercise alone experiments. Those results suggest that adenine nucleotide degradation enhanced by both beer ingestion and muscular exercise, lactic acid production from muscular exercise, a slow decrease in blood concentration of lactic acid during ethanol metabolism, and decreased creatinine clearance from exercise together cause a synergistic increase in the plasma concentration of uric acid. Therefore, exercise is a factor involved with the acceleration of ethanol-induced increase in plasma concentration of uric acid. As for oxypurines, it was demonstrated that an increase in plasma concentration and urinary excretion of xanthine in our combination experiment was less than that in the beer ingestion experiment, though the increases in plasma concentration of hypoxanthine were not different between the combination and beer ingestion experiments. In addition, we found that the blood concentration of pyuvic acid was not changed in the combination experiment, whereas it decreased in the beer ingestion experiment. These results suggest that the small increases in plasma concentration and urinary excretion of xanthine in the combination experiment were due to a relief of the ethanol-induced

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inhibition of xanthine dehydrogenase activity, since pyruvic acid produced by exercising muscles may be partly transported to the liver and metabolized to lactic acid, coupled with a conversion of NADH to NAD, thereby decreasing in the ratio of NADH/NAD. Further, we found that NH 3 produced by the deamination of AMP in exercising muscles was increased in the exercise and combination experiments, suggesting that the ethanol-induced inhibition of xanthine dehydrogenase activity was relieved by increased NH3, since produced NH3 is transported to the liver and removed through the mediation of glutamate dehydrogenase to form glutamate and NAD from 2-oxyglutarate, NH3, and NADH. 6.3. Dehydration (decreased extracellular volume) Following excessive perspiration, dehydration can result in increased plasma concentration [1]. The resulting decreased extracellular volume also contributes to decreased fractional excretion of uric acid. Ethanol ingestion induces diuresis, leading to dehydration, which is a factor for increased plasma uric acid level, as described above. However, the effect of a combination of excessive perspiration and ethanol ingestion on the plasma concentration of uric acid has not been examined, though it is thought to be clinically important. Recently, we examined the effect of dehydration caused by sauna bathing on the beerinduced increase in plasma concentration of uric acid [115]. Sauna bathing led to decreased body weight of approximately 800 g and a slightly increased plasma concentration of uric acid by 19 Amol/L (0.3 mg/dL) together with a decrease in the urinary excretion of uric acid by 39%, while a combination of beer ingestion (10 ml/kg body weight) and sauna bathing led to an increase in plasma concentration of uric acid by 0.64 Amol/L (1.1 mg/dL). However, the combination did not severely affect the urinary excretion of uric acid, since beer contains enough fluids to transiently relieve dehydration. Those results suggest that the effect of beer ingestion just after sauna bathing is somewhat additive, since beer ingestion increased the plasma concentration of uric acid by 0.34 Amol/L (0.57 mg/dL). By relieving the dehydration-induced decrease in urinary excretion of uric acid, the effect of a combination of beer and sauna bathing on the plasma concentration of uric acid was

reduced. However, since ethanol decreases the secretion of ADH, dehydration, such as that caused by sauna bathing, may enhance the ethanol-induced increase in plasma concentration of uric acid. 6.4. Drugs (pyrazinamide, furosemide) Pyrazinamide is an antituberculosus agent that is frequently used for the treatment of tuberculosis and its metabolite, pyrazinoate, markedly decreases the urinary excretion of uric acid, which has been attributed to inhibition of the secretory component of bidirectinal uric acid transport in the proximal tubule [116]. Previously, Guggino et al. showed that low doses of pyrazinoate stimulated uric acid reabsorption across the luminal membrane of proximal tubular cells [117]. In addition, Enomoto et al. recently demonstrated that pyrazinoate was transported from tubular cells into the tubular lumen by URAT1 in vitro, in combination with the transport of uric acid from the tubular lumen into tubular cells, as in the case of lactic acid (Fig. 11) [83]. Those findings strongly suggest that pyrazinoate stimulates uric acid reabsorption via URAT1. In any case, marked hyperuricemia often develops in subjects receiving pyrazinamide. Therefore, it is suggested that ingestion of alcoholic beverages may synergistically increase the plasma concentration of uric acid. Furosemide is a diuretic used for the treatment of heart failure, hypertension, and edema. It inhibits the absorption of chloride and sodium at Henle’s loop, resulting in an increase in urine formation together with natriuresis [118]. Thiazide is also a diuretic that is used for the treatment of hypertension, which inhibits the absorption of chloride and sodium at the distal tubules, resulting in an increase in urine formation together with natriuresis [119]. These effects lead to a decrease in extracellular fluid volume, which causes a decrease in the clearance of uric acid. As a result, hyperuricemia develops. From those findings, it is suggested that these diuretics may aggravate ethanol-induced hyperuricemia. 6.5. Others Many other factors, such as obesity, hyperinsulinemia, purine-rich foods and beverages, may indi-

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rectly contribute to acceleration of the ethanolinduced increase in the plasma concentration of uric acid, though they seem to play a minor role. 6.5.1. Obesity Obesity is a factor known to increase the plasma concentration of uric acid [31]. Recently, obesity was classified into visceral fat and subcutaneous fat types. Visceral fat obesity seems to be related to uric acid metabolism [29,120] and insulin resistance (hyperinsulinemia) [121]. Takahashi showed that visceral fat accumulation was negatively correlated with uric acid clearance and Facchini et al. reported that urinary uric acid clearance appeared to decrease in proportion to increases in insulin resistance in normal volunteers, leading to an increase in serum uric acid concentration. Those findings suggest that visceral fat accumulation leads to insulin resistance, due to increased free fatty acid, increased TNFa, and decreased adiponectin, leading to a decrease in urinary uric acid clearance, which causes hyperuricemia. Further, visceral fat obesity seems to be related to an increased urinary excretion of uric acid (an increased production of uric acid), though its mechanism remains unknown [29,120]. Accordingly, obesity, especially visceral fat obesity, may accelerate the ethanol-induced increase in plasma concentration of uric acid. 6.5.2. Ingestion of purine-rich foods Purine-rich food consumption is known to raise the plasma concentration of uric acid [122,123], and eating purine-rich meat and seafood has been associated with an increased risk of gout [124]. Alcoholic beverages are frequently ingested together with purine-rich foods and, since purine-rich food intake causes an increase in serum uric acid levels within about 2 h [122,123], it may accelerate the ethanolinduced increase in serum concentration of uric acid, such as the purines present in beer.

7. Prevention and treatment of ethanol-induced hyperuricemia 7.1. Restriction of ingested ethanol volume Several studies [5,45,46] have confirmed the association between alcohol consumption and risk of

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gout. In addition, in many reports [8–10,13,19,20,22, 52,54–56] of the effects of alcohol ingestion on the plasma concentration of uric acid and/or oxypurines, a rough correlation between the blood concentration of ethanol and increase in plasma concentration of uric acid have been shown. Therefore, it is clinically important to restrict ingestion of all types of alcoholic beverages to prevent the onset of hyperuricemia and gouty attack. Although it remains undetermined to what extent should be restricted, the relative risk for incidence of gout according to alcohol intake is shown in Table 1. 7.2. Restriction from purine-containing alcohol beverages Alcoholic beverages have a variety of effects on the increase in plasma concentration of uric acid. In a recent study [47], ingestion of two or more beers (710 ml or more) per day increased the risk of gout by 2.5fold, as compared with no beer intake, whereas two or more shots of spirits (88 ml or more) per day increased by 1.6 times, as compared with no intake. Beer increased the risk of gout per serving each day by more than twice as much as regular spirits (49% vs. 15%), even though ethanol content per serving was less for beer than regular spirits (12.8 vs. 14.0 g). Those results suggest that some non-alcoholic components in beer, presumably purines, play an important role in the incidence of gout, as beer contains more purines than other beverages, such as regular spirits and wine. Since the purines in beer are converted to uric acid in the body, the plasma concentrations of uric acid and oxypurines may increase at the blood ethanol level of 30 to 50 mg/ dL (6.5–10 mmol/L), at which other beverages that contain few purines do not increase the plasma concentration of uric acid. In fact, purine-free happou-shu did not increase the plasma concentration of uric acid, while regular happo-shu did, though the ingested ethanol volume was the same [106]. However, since an increase in the ingested volume of purine-free happo-shu raises the plasma concentration of uric acid, purine-free happo-shu seems to be better than other beverages for plasma uric acid only within a range of 6.5–10 mmol/L of blood ethanol. Further, though beer seems to affect the plasma concentration of uric acid to an extent greater than other alcoholic

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beverages at the same blood ethanol level, it remains undetermined whether it is clinically important to change beer ingestion to other beverages. In contrast to beer, drinking less than 236 ml (less than 22.0 g ethanol) of wine per day did not increase the risk of gout in a recent prospective study [47], which suggested that the non-alcoholic components in wine may be protective factors against gout. However, ingestion of red wine (5 ml/kg body weight) rapidly increased serum uric acid [125], while four glasses of red wine each day for 12 weeks increased the serum concentration of uric acid by 9%, as compared to 8% by regular spirits. These findings suggest that the effect of red wine on the serum concentration of uric acid is not significantly different from that of regular spirits. Thus, red wine does not seem to have advantages over other beverages in terms of the effect on uric acid. 7.3. Change in drinking habits A previous study demonstrated that habitual alcohol drinking accelerates ethanol metabolism and, thereby, may enhance ethanol-induced adenine nucleotide degradation, suggesting that a drinking habit is a factor for the acceleration of the ethanolinduced increase in plasma concentration of uric acid [20]. Therefore, it is recommended that ingestion of alcoholic beverages be decreased in terms of frequency as well as volume, as much as possible, by patients with gout. 7.4. Weight control, restriction of purine-rich and high calorie food, restriction of rigorous exercise, and prophylaxis for dehydration Hyperuricemia is ascribable to many factors, which can be classified into genetic and environmental factors including drinking habit, high purine intake, and obesity due to excessive calorie intake, as well as others. The onset of hyperuricemia depends on the influence of genetic and/or that of environmental factors. Although genetic factor seems to be more important, others also play a role in the onset of hyperuricemia. Therefore, to protect against the onset of ethanol-induced hyperuricemia, restrictions against excessive calorie intake, high purine foods, dehydration, and excessive muscular exercise, especially

anaerobic exercise, as well as drinking alcoholic beverages, are needed. 7.5. Administration of anti-hyperuricemic agents (allopurinol and benzbromarone) Allopurinol is an important anti-hyperuricemic agent and many patients with gout throughout the world have been treated with this drug. It is a potent xanthine dehydrogenase (xanthine oxidase) inhibitor that lowers the plasma concentration of uric acid and may play an important role for the treatment of alcohol-induced hyperuricemia. However, in a previous study it was demonstrated that the intake of allopurinol together with whiskey decreased the plasma concentration of oxypurinol, a metabolite of allopurinol, as compared with allopurinol alone (Fig. 6). Oxypurinol is also a potent inhibitor of xanthine dehydrogenase and its biological half-life (about 18 h) is longer than that of allopurinol (about 4 h) [126– 129]. Therefore, the overall effect of allopurinol on the plasma concentrations of purine bases is mostly dependent on oxypurinol. Although the decrease in plasma concentration of oxypurinol following whiskey ingestion seems to depend on the slight inhibition of xanthine dehydrogenase by ethanol metabolism, it may be clinically important, since the effect of allopurinol on plasma uric acid may also be decreased by whiskey. Therefore, it is recommended that allopurinol be taken more than 4 h prior to alcohol ingestion. Uricosuric agents (benzbromarone, probenecid) also seem to be effective for the control of ethanol-induced hyperuricemia. However, since the intake of benzbromarone together with whiskey decreases the plasma concentration of benzbromarone, as compared with that of benzbromarone alone, the effect of bezbomarone on the plasma concentration of uric acid may be reduced, though the mechanism is unknown. Therefore, for gout patients with a daily drinking habit attention must be paid to the administered dose of benzbromarone. In addition, since patients with heavy drinking habit tend to forget to take medicine needed for their treatment, it is important to confirm that gout patients who drink heavily continue to take antihyperuricemic agents in order to lower the plasma concentration of uric acid.

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8. Summary The effects of alcoholic beverages on the plasma concentrations and urinary excretion of purine bases were reviewed especially in terms of epidemiology, mechanism, prevention, and treatment. However, since many factors associated with the influence of ethanol on purine bases remain unknown, further study is needed to accurately elucidate those effects.

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