Carotenoids: Liver Diseases Prevention
Oxidative Stress and Carotenoids
Oxidative Stress and Liver Diseases
The liver is a vital organ and has a wide range of functions, including detoxification, protein synthesis, production of biochemicals necessary for digestion, and maintenance of normal glucose concentrations during fasting. This organ plays an important role in the metabolism of glycogen storage, plasma protein synthesis, hormone production, and detoxification. Especially, the liver is a major site of insulin clearance and the loss of a direct effect of insulin to suppress hepatic glucose production, and glycogenolysis in the liver causes an increase in hepatic glucose production (Michael et al., 2000).
Chronic liver disease is a worldwide common pathology. Abnormal liver function is characterized by an inflammatory and fibrotic process that leads to a progressive evolution from chronic hepatitis to cirrhosis and liver cancer. Alcohol, virus, xenobiotics, and/or unusual lipid and carbohydrate metabolism such as obesity, insulin resistance, and type 2 diabetes are widely known causes of chronic liver disease (Loguercio et al., 2001).
Oxidative stress plays a major role in the pathogenesis of liver injuries. The major source of reactive oxygen species in the liver are the activated inflammatory cells, such as macrophage and Kupffer cells, the mitochondrial enzymes, and cytochrome P450 of damaged liver cells. The excessive reactive oxygen species in the liver affects not only the transcription of biochemical mediators such as cytokines which modulate tissue and cellular events but also degeneration of lipids, proteins, DNA, carbohydrates, and other biomolecules (Parola and Robino, 2001; Tilg and Diehl, 2000). In such circumstances, antioxidant micronutrients, such as carotenoids, may play important roles in defending against oxidative stress by efficiently quenching the production of singlet oxygen and free radicals and inhibiting the progression of liver diseases.
Role of Antioxidant Carotenoids
Antioxidant micronutrients, such as vitamins and carotenoids, exist in abundance in fruit and vegetables and have been known to contribute to the body’s defense against reactive oxygen species (Stanner et al., 2004). Recently, it became known that antioxidant vitamins and carotenoids are reduced in several liver diseases, such as hepatitis and cirrhosis (Jain et al., 2002; Leo et al., 1993; Van de Casteele et al., 2002; Ward and Peters, 1992). Oxidative stress is thought to play a key role in the pathogenesis of liver injury. Therefore, antioxidant carotenoids would be expected to protect against liver injury.
Ingested carotenoids from foods exist in several human organs (Stahl et al., 1992). Carotenoids are mainly accumulated in the liver and combined into lipoprotein for release into the blood circulation. Ingested carotenoids could participate in an antioxidant defense system when present in high concentrations of free radical species in the liver, and these physiological functions of carotenoids could inhibit the development of liver dysfunction. In fact, recently, many studies have been reported that carotenoids, such as b-carotene, lycopene, lutein, and b-cryptoxanthin, have antioxidant effects against lipid peroxidation in rat liver (Chen and Tappel, 1996; Whittaker et al., 1996).
Alcoholic Liver Diseasea and Carotenoids
It is well known that alcohol induces the generation of free radical species during its metabolism (Koch et al., 2004). The absorbed ethanol is oxidized to acetaldehyde by acetaldehyde dehydrogenase in mitochondria. In habitual drinkers, the microsomal ethanol-oxidizing system is increased by enzyme induction and is also responsible for the production of acetaldehyde. The generation of high concentrations of free radical species during the metabolism of alcohol may exceed the capacity of the antioxidant defense mechanisms and cause the development of liver dysfunction.
Clinical Case–Control Studies
Many studies of the antioxidant status of alcohol-induced hepatitis or cirrhosis patients have reported on the measurement of the blood concentrations of antioxidants or markers of oxidative stress, such as a-tocopherol, ascorbic acid, carotenoids, or glutathione (Leo et al., 1993; Van de Casteele et al., 2002; Ward and Peters, 1992).
Ward and Peters (1992) examined the plasma antioxidant values of alcoholic patients. They found that the alcoholic patients’ group showed significant decreases in the mean plasma values of b-carotene, zinc, and selenium when compared to the control subjects. When the patients were subdivided according to their liver histology, b-carotene showed a progressive decrease in plasma concentration with increasing liver damage, whereas a-tocopherol levels were only depleted in the patients with cirrhosis.
Similarly, Van de Casteele et al. (2002) also have investigated whether various antioxidant parameters in blood are affected in different stages of alcoholic liver disease and how specific the changes are relative to nonalcoholic cirrhosis. In this study, patients with alcohol abuse without cirrhosis, with alcoholic cirrhosis, and with nonalcoholic cirrhosis stratified by Child–Pugh scores (A, B, and C) were studied. The Child–Pugh score is used to assess the prognosis of chronic liver disease, mainly cirrhosis. Levels of reduced glutathione and glutathione peroxidase activity in blood, erythrocytic superoxide dismutase activity and carotenoids, a-tocopherol and malondialdehyde in plasma were measured. As a result, they found that levels of reduced glutathione were significantly decreased in Child–Pugh score C cirrhotics, alcoholic or not in origin, whereas oxidized glutathione and glutathione peroxidase activity were not affected. Superoxide dismutase activity and a-tocopherol levels were not significantly different in the various groups. In contrast, total carotenoid levels (a-carotene, b-carotene, lycopene, cryptoxanthin, lutein, and zeaxanthin) were significantly lower in alcoholic cirrhotics (Child–Pugh score C) versus controls. Malondialdehyde levels were elevated only in cirrhotics’ Child–Pugh score C, alcoholic or nonalcoholic.
On the other hand, Leo et al. (1993) examined the carotenoid levels in diseased liver. In this study, they measured six carotenoid contents in diseased liver of patients with alcoholic cirrhosis, less severe alcoholic liver disease, and with nonalcoholic liver disease and compared them with control subjects. As a result, they found that all carotenoid levels were extremely low at all stages of liver disease. Patients with alcoholic cirrhosis had 20- and 25-fold decreases of levels of lycopene and a- and b-carotene, respectively. Even in subjects with less severe alcoholic liver disease (steatosis, perivenular fibrosis, and portal fibrosis) and in patients with nonalcoholic liver disease, levels were four to six times lower than those in normal subjects.
These clinical case–control studies provided support for the hypothesis that antioxidant carotenoids may protect against oxidative stress induced by alcohol consumption.
Observational Epidemiological Studies
Serum liver enzymes with carotenoids
In contrast, some observational epidemiologic studies on normal living subjects about the associations of serum carotenoid concentrations and liver functions have been reported. In these epidemiologic studies, investigators measured serum liver enzyme activities in the blood. The damage of liver cells accompanies the release of liver function enzymes, such as g-glutamyltransferase (g-GTP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) into the blood. Clinically, these enzymes are often used as biomarkers for liver injury or liver diseases. Recently, several epidemiologic studies have shown that serum g-GTP is associated with risk factors of cardiovascular disease, suggesting the possibility of a close relationship between oxidative stress and chronic diseases (Lee et al., 2004; Nakanishi et al., 2003).
g-GTP exists widely in various tissues, especially in the liver and kidneys, and it catalyzes the transfer of a g-glutamyl group from g-glutamyl peptides to other peptides, thereby providing a supply of constituent amino acids for uptake and reutilization in intracellular glutathione synthesis. In a normal metabolism, this enzyme plays an important role in antioxidant defense systems on a cellular level. Although serum g-GTP is not a specific indicator of liver injuries due to alcohol, it is widely used as a screening test for alcohol-induced liver dysfunction (Ryback et al., 1982).
A recent epidemiological study has reported the inverse association of serum g-GTP with serum-carotenoid concentrations both cross-sectionally and longitudinally (Lee et al., 2004). In this study, the inverse associations between total serum carotenoid concentration and serum g-GTP were examined among subgroups including race, sex, body mass index (BMI) levels, tobacco use, and vitamin supplement usage. With regard to alcohol consumption, although inverse associations between total serum carotenoid concentration and serum g-GTP were found in drinkers, they were not found in nondrinkers. However, the associations of the serum carotenoid concentration and serum g-GTP with ethanol intake were not discussed in detail according to the stratification of the ethanol intake level. Furthermore, the associations of serum concentration of each carotenoid with serum g-GTP were not explained in detail.
Alcohol-induced increases of serum liver enzymes with carotenoids
On the other hand, Sugiura et al. (2005) examined the associations of six main serum carotenoids and serum g-GTP with alcohol intake level. In this study, the daily ethanol questionnaire concerning food frequency. Therefore, these data make it possible to evaluate the detailed association of serum carotenoid concentrations and serum g-GTP with alcohol intake level. They evaluated the association of serum g-GTP as a marker of oxidative stress induced by alcohol consumption and serum carotenoids stratified by alcohol intake level in Japanese men with normal liver function.
The subjects were divided into three groups stratified by ethanol intake levels defined as nondrinkers (less than 1 g of ethanol daily), light drinkers (#1, <25 g of ethanol daily), and moderate and heavy drinkers (#25 g of ethanol daily). The subjects were further subcategorized into three groups according to the tertile of serum carotenoid concentrations after being stratified by ethanol intake levels. The multivariate-adjusted geometric mean and 95% confidence interval of the serum g-GTP concentrations by tertiles of the serum carotenoid concentration were calculated after adjusting for confounders using analysis of covariance.
In light drinkers, the adjusted means of serum g-GTP were slightly low in accordance with tertiles of serum b-carotene and b-cryptoxanthin concentration but the group difference was not statistically significant. In moderate and heavy drinkers, adjusted means of serum g-GTP were significantly low in accordance with the tertiles of serum lycopene, a-carotene, b-carotene, and b-cryptoxanthin. On the other hand, significant inverse associations were not observed in lutein and zeaxanthin.
These results suggest that carotenoids may act as a suppressor against liver cell damage and inhibit the progression of liver dysfunction induced by alcohol. However, the data obtained here consist of cross-sectional analyses. To determine whether serum antioxidant carotenoids are effective in increasing serum g-GTP in alcohol drinkers, further cohort studies or intervention studies will be required.
Cigarette Smoking Exacerbates Depletion of Serum Carotenoids Induced by Alcohol?
As mentioned earlier, alcohol induces the generation of free radical spices during metabolism in the liver. In such circumstances, carotenoids may play important roles as antioxidants in defending against oxidative stress. That is to say, carotenoids may be consumed by free radical species. On the other hand, active smokers are exposed to reactive free radicals that are present in cigarette smoke. Therefore, smoking is also a potent oxidative stress in humans. However, there is limited information about the synergistic interaction of cigarette smoking and alcohol drinking with serum carotenoid concentrations. The differences in the change among six major serum carotenoid concentrations against oxidative stress induced by cigarette smoking and alcohol drinking have not been thoroughly studied while controlling for dietary carotenoid concentrations.
Synergistic interaction of cigarette smoking and alcohol drinking with serum carotenoids
Very recently, Sugiura et al. (2009) tested the hypothesis that smoking and drinking reduce the serum carotenoid concentration synergistically. In this survey, the subjects were divided into six groups according to alcohol intake (nondrinkers, <1 g per day; light drinkers, #1, <25 g per day; moderate to heavy drinkers, #25 g per day) and smoking status (nonsmokers and current smokers). The dietary intakes and serum concentrations of six carotenoids (lycopene, a-carotene, b-carotene, lutein, b-cryptoxanthin, and zeaxanthin) within each group were evaluated cross-sectionally. Results showed that the multivariate-adjusted means of the serum carotenoid concentrations in nondrinkers did not differ between nonsmokers and current smokers. In contrast, the adjusted means of serum a-carotene, b-carotene, and b-cryptoxanthin were significantly lower than those with increased alcohol intake, and these lower serum carotenoids among alcohol drinkers were more evident in current smokers than in nonsmokers. Serum lycopene of moderate to heavy drinkers was significantly lower than that of nondrinkers, but it was not influenced by smoking. Neither smoking nor drinking was associated with the serum concentrations of lutein and zeaxanthin. These differences of serum carotenoid concentrations among six groups were observed after adjusting for intakes of each carotenoid. From this study, it was observed, interestingly, that serum a-carotene, b-carotene, and b-cryptoxanthin in moderate to heavy drinkers among current cigarette smokers were about half the levels of those in nondrinkers among nonsmokers even though their intake of carotenoids was the same. These results suggest that smoking and drinking may reduce the serum a-carotene, b-carotene, and b-cryptoxanthin concentrations in a synergistic manner.
Differences among six carotenoids against oxidative stress induced by cigarette smoking and alcohol drinking
It is known that a transforming reaction from pro-vitamin A to retinol is induced by smoking. On the other hand, alcohol is known to promote increased oxidation of vitamin A compounds and reduce liver stores. It can be postulated that alcohol intake may also accelerate the conversion of pro-vitamin A to retinol. From among the six major serum carotenoids, a-carotene, b-carotene, and b-cryptoxanthin, are pro-vitamin A. These three carotenoids are converted to retinol in the body. Therefore, the serum concentrations of a-carotene, b-carotene, and b-cryptoxanthin might be more easily influenced by cigarette smoking and alcohol drinking than lycopene.
Sugiura et al. (2009) also found that the serum lutein and zeaxanthin concentrations were not influenced not only by alcohol drinking but also by cigarette smoking. It might be difficult to expose lutein and zeaxanthin against oxidative stress or that the differences among the six serum carotenoids observed occurred by chance. One possible explanation is that the differences in the associations of the six serum carotenoids with cigarette smoking and alcohol drinking might be attributed to the polar characteristics of each carotenoid. It is conceivable that the tissue distribution and localization in the cell membranes of carotenoids differ in each carotenoid. Especially, the chemical structure of a carotenoid may determine its localization in a cell membrane. Hydrocarbon carotenoids, such as lycopene, a-carotene, and b-carotene, are located within the hydrophobic membrane core with multiple orientations, whereas xanthophylls, such as lutein and zeaxanthin, have a more rigid membrane-spanning orientation. Therefore, the antioxidant defense system by carotenoids against lipid peroxidation in a cell membrane may depend on the polar characteristics of each carotenoid.
Nonalcoholic Liver Disease and Carotenoids
The adverse impact of overweight, obese, and/or physical inactivity is well documented as risk factors for diabetes, cardiovascular disease, cancer, and musculoskeletal disease. Nonalcoholic fatty liver disease (NAFLD) presents a comprehensive histological aspect which results from the deposit of triglycerides into hepatocytes. Although the pathological alterations in NAFLD are similar to those of alcoholic liver disease, it occurs in nonalcoholic individuals. These pathological alterations vary from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. The prevalence of NAFLD has risen in parallel with obesity and diabetes, and it is becoming the most common cause of liver disease in Western countries.
Nonalcoholic Liver Disease and Oxidative Stress
The role of oxidative stress and mitochondrial dysfunctions in nonalcoholic liver diseases is well documented (Basaranoglu et al., 2010). b-Oxidation within the normal liver takes place in mitochondria, but this process in the context of NAFLD can become overwhelmed as a result of increased free fatty acid (FFA) load. Increased FFA gives rise to reactive oxygen species. Reactive oxygen species induce oxidative stress and activate inflammatory pathway. Although antioxidant carotenoids may also act as suppressors to oxidative stress in nonalcoholic liver diseases, the associations of carotenoids with nonalcoholic liver diseases have not been thoroughly studied.
Relationship Between Serum Liver Enzymes and Carotenoids Associated with Nonalcoholic Liver Disease
One large observational epidemiologic study has been reported about the inverse association of serum carotenoid concentrations with abnormal serum liver enzyme activity. Ruhl and Everhart (2003) analyzed the associations of serum antioxidants with abnormal serum ALT activity from the third US National Health and Nutrition Examination Survey (NHANES III) using a total of 13,605 adult subjects. According to results, they found that abnormal ALT risk was associated negatively with an increase of a-carotene, b-carotene, b-cryptoxanthin, lutein/zeaxanthin, and combining 5 carotenoids. They also examined the associations of serum carotenoid concentration with the risk for abnormal ALT level among subcategorized subjects by obesity, waist-to-hip ratio, and diabetes. Significant inverse association of combined 5 carotenoids with the risk for abnormal ALT level was observed among obese subjects (BMI, #25) but not among non-obese subjects. The same inverse association was also observed among subjects whose waist-to-hip ratio was higher (#0.87) but not among normal subjects. From these results, one may conclude that carotenoids may act as suppressors against abnormal liver function such as nonalcoholic liver disease associated with visceral adiposity.
Hyperglycemia-Induced Increases of Serum Liver Enzymes with Carotenoids
On the other hand, chronic hyperglycemia leads to the auto-oxidation of glucose and causes the nonenzymatic glycation of proteins through Maillard’s reaction (Giugliano et al., 1996). In these processes, reactive oxygen species are produced. Hyperglycemia enhances oxidative stress, for example, the increase of lipid peroxidation. The relationship between the pathogenesis of diabetes mellitus and oxidative stress is unclear, but a close relationship exists between the pathogenesis of diabetic complications and tissue injury from free radicals. To avoid oxidative stress, antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, play an important role against oxidative stress. However, the generation of high concentrations of free radical species in hyperglycemia also causes nonenzymatic glycation of these antioxidant enzymes (Vijayalingam et al., 1996). These facts indicate that hyperglycemia-induced oxidative stress may also cause liver cell damage, and increased oxidative stress in hyperglycemia causes an increase in serum liver enzyme even at physiological concentrations. Therefore, an antioxidant defense system against oxidative stress induced by chronic hyperglycemia may play an important role in the earlier pathogenesis of liver disease among hyperglycemic subjects. If so, carotenoids may act as suppressors to inhibit the progression of liver disease induced by hyperglycemia, and this will eventually result in the prevention of nonalcoholic liver disease.
Recently, Sugiura et al. (2006) examined the hypothesis that hyperglycemia-induced increases of serum liver enzymes at physiological concentrations would be inversely associated with the serum carotenoid concentrations. The associations of the six serum carotenoid concentrations with serum liver enzyme stratified by the glucose tolerance status were evaluated cross-sectionally.
AST and ALT serum liver enzyme concentrations in the impaired fasting glucose (IFG) and diabetes groups were significantly higher than those in the normal fasting glucose (NFG) group. The means of the serum ALT concentrations in each tertile were calculated after adjusting confounders. IFG and diabetic groups were combined to form a hyperglycemic group. The serum AST concentration in the hyperglycemic group was significantly low in accordance with the tertiles of serum b-carotene and b-cryptoxanthin concentrations. On the other hand, serum ALT concentration in the hyperglycemic group was significantly low in accordance with the tertile of the serum b-cryptoxanthin concentration.
These results showed that serum b-carotene and b-cryptoxanthin concentrations were inversely associated with serum AST and ALT concentrations in the hyperglycemic subjects. The inverse associations of serum carotenoid concentrations, especially in b-cryptoxanthin, with serum AST and ALT were progressively stronger in glucose intolerance. b-Carotene and b-cryptoxanthin may act as a suppressor against liver cell damage and inhibit the progression of liver dysfunction in hyperglycemia.
Liver Cancer and Carotenoids
Although the potential roles of antioxidant carotenoids in cancer prevention have been demonstrated at various cancer sites (Druesne-Pecollo et al., 2010), the association with hepatocellular carcinoma (HCC) remains unclear (World Cancer Research Fund/ American Institute for Cancer Research, 2007). Recently, some large cohort studies about the association of carotenoid intake with the risk of liver cancer and an intervention study were reported.
Large Cohort Study
Kurahashi et al. (2009) have reported the inverse associations of intakes of green vegetables or carotenoids with the risk for HCC using 19,998 men and women (235,811 person-years of follow-up). As a result, they found that borderline inverse associations were seen between total vegetables and green-yellow vegetables and HCC, with multivariable hazard ratios (HRs) for the highest versus the lowest tertile of 0.61 (95% confidence interval (CI) ¼ 0.36–1.03, P for trend ¼ .07) and 0.65 (95% CI ¼ 0.39–1.08, P for trend ¼ .06), respectively. In particular, green leafy vegetable consumption showed an inverse dose-dependent association with HCC (HR ¼ 0.59, 95% CI ¼ 0.35–1.01 for highest versus lowest tertile of consumption, P for trend ¼ .04). On the other hand, a slightly negative association was seen between a- and b-carotene and HCC, with respect to multivariable HRs for the highest versus the lowest tertile of 0.69 (95% CI ¼ 0.42–1.15) and 0.64 (95% CI ¼ 0.38–1.08). Furthermore, they found that these inverse associations of vegetable and carotenoid intakes with the risk for HCC were noted especially among those who were never cigarette smokers (highest versus lowest: HR ¼ 0.31, 95% CI ¼ 0.13–0.76 for b-carotene).
Meanwhile, Nishino et al. (2009) examined the effectiveness of combinational administration of multiple carotenoids against liver cancer in hepatitis virus-infected patients with cirrhosis. In the past, they found that palm oil carotene, which consists of 30% a-carotene, 60% b-carotene, and 10% others (g-carotene, lycopene, etc.), remarkably suppressed spontaneous liver carcinogenesis in C3H/He male mice, more effectively than a- or b-carotene alone (Murakoshi et al., 1992). Therefore, they examine the hypothesis that a carotenoid mixture would be effective to suppress the development of liver cancer in patients with hepatitis C virus-induced liver cirrhosis, the high risk group of HCC. As a result, they found that the administration of hydrocarbon carotenoid mixture (lycopene 10 mg, b-carotene 6 mg, and a-carotene 3 mg) resulted in significant suppression of tumor development. Furthermore, recently, they found that combined application of Japanese mandarin orange juice containing 3 mg of b-cryptoxanthin with a carotenoid mixture (lycopene 10 mg, b-carotene 6 mg, and a-carotene 3 mg) showed to be more effective than a carotenoid mixture alone.
From these results, various carotenoids and combinations of these elements seem to be promising for the prevention of a wide variety of chronic liver diseases, not only liver cancer but also alcoholic and nonalcoholic diseases, although further extended clinical trial is needed to confirm and improve the efficacy.
Systemic inflammation and oxidative stress appear to be involved in the progression of liver dysfunction. Alcoholic and nonalcoholic fatty liver diseases are conditions associated with higher levels of inflammatory proteins, increased markers of oxidative stress, and lower plasma concentrations of antioxidants. On the other hand, prospective cohort studies have linked the consumption of fruit and vegetables to a decreased risk of liver disease, cardiovascular events, metabolic syndrome, and type 2 diabetes, which suggests a protective effect of dietary antioxidant carotenoids. Indeed, public health authorities have been recommended a diet of five servings of fruit and vegetables each day. Fruit and vegetables are the main dietary sources of carotenoids. Ingested carotenoids could participate in an antioxidant defense system when present in high concentrations of free radical species in the liver, and these physiological functions of carotenoids could inhibit the development of liver dysfunction. However, even if antioxidant carotenoids are thought to play a key role in disease prevention, the results of intervention studies with single antioxidants administered as supplements have been poor so far. The consumption of carotenoids in pharmaceutical forms for the treatment or prevention of these chronic diseases cannot be recommended, because some large randomized controlled trials did not reveal any reduction in cancer, cardiovascular events, and/or type-2 diabetes with b-carotene (Liu et al., 1999; To¨rnwall et al., 2004). High doses of carotenoids used in the supplementation studies could have a pro-oxidant effect (El-Agamey et al., 2004). Therefore, it might be favorable to consume carotenoids from foods through the combination of other nutrients such as vitamins, minerals, or phytochemicals, and not by supplements.
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