Introduction

l-Methionine is an essential, non-polar amino acid that is usually obtained from outside sources such as sesame oil, meat, and fish (Martinez et al. 2017). l-Methionine is considered as intermediate substrate for the synthesis of other amino acids such as homocysteine, which is formed by demethylation of l-methionine (Cui et al. 2012; Lee and Gladyshev 2011).

Homocysteine is a non-essential amino acid that is already present in the blood. It has been implicated in oxidative stress and cognitive dysfunction (Bhargava et al. 2018). Recent studies on animals have shown that chronic administration of l-methionine results in hyperhomocysteinemia (Kumar et al. 2017), and the subsequent development of cerebrovascular diseases, including stroke (Azad et al. 2018), atherosclerosis (Aronow 2003; Cacciapuoti 2013), and vascular dementia (Bonetti et al. 2016). In addition, hyperhomocysteinemia is associated with increased levels of oxidative stress and lipid peroxidation that interfere with memory formation (Cheng et al. 2016; Kolling et al. 2017; Zeng et al. 2018). Hyperhomocysteinemia also contributes to the increased risk of dysfunction in endothelial cells that may interfere with blood supply to the brain and the subsequent cognitive decline (Koladiya et al. 2008).

Caffeine (1,3,7-trimethylxanthine) is a non-specific adenosine antagonist and a psychoactive stimulant. It is widely consumed and naturally produced from the leaves and seeds of many plants such as tea and coffee (Grosso et al. 2017). Studies showed that caffeine has antioxidant effect in circulating blood (Jasiewicz et al. 2016; Onaolapo et al. 2016; Viollet et al. 2012) and has efficacy to reduce serum lipids (Millar et al. 2017). In the current study, the possible preventive effect of caffeine against impairment of memory induced by l-methionine chronic administration was examined. The results might be useful in the management of adverse health effects of l-methionine consumption on body health.

Material and Methods

Wister rats (adult, males) weighing 200–220 g were housed in metallic cages (2–5 rats in each cage) under hygienic conditions, at room temperature, and given ad libitum access to tap water and food. The animals were housed on a 12/12-h light/dark cycle with the light period starting at 7:00 p.m. The rats were given 1 week of acclimation before the experimental procedures began. The study procedures were approved by Institutional Animal Care and Use Committee on June 01, 2011.

Animals were randomly assigned to four groups: control (Control), l-methionine (l-Meth), caffeine (Caf), and l-methionine with caffeine (l-Meth+Caf). l-Methionine and caffeine were purchased from Sigma Chemical Co. (Saint Loius, MO, USA). l-Methionine (1.7 g/kg/day, suspended in 0.5% w/v carboxylethyl cellulose (CMC)) was administered by oral gavage (Alzoubi et al. 2014) while caffeine (0.3 g/L) was supplemented in drinking water (Alhaider et al. 2010; Alzoubi et al. 2013a, c). Animals in the Control and Caf groups were administered the vehicle via oral gavage (0.5% w/v CMC). All manipulations including administration of l-methionine, caffeine, and vehicle were started on the same day, and continued for 4 weeks. The RAWM training was carried out immediately after treatment. Administration of drugs was continued throughout the radial arm water maze (RAWM) testing day.

The Radial Arm Water Maze (RAWM)

Spatial learning and memory was tested using RAWM (Alzoubi et al. 2016, 2018; Mhaidat et al. 2015; Rababa'h et al. 2017). The RAWM consists of six swimming paths protruding out of an area at the center. There is also a hidden platform located at the end of one of the six arms (the goal arm). The water was maintained at 24 ± 1 °C. The room where the experiments were carried out was dimly lit with two different patterns on the walls, which served as visual cues for the rats. The animals have to find the hidden platform, which was not changed for a particular rat. Rats were given six consecutive trials, separated a rest of 5 min, followed by another six successive trials (the learning phase), then a short-term memory test at 30 min and two long-term memory tests at 5 h and 24 h. The RAWM procedure was done as previously described (Alzoubi et al. 2013a, b).

Animals’ Subtotal Exsanguinations and Brain Dissection

The animals were anesthetized using 40 mg/kg of thiopental administered intraperitoneally (IP); thereafter, animals were killed using subtotal exsanguinations. Then, the brain was dissected out; the hippocampus was removed and immediately frozen using liquid nitrogen.

Calorimetric Immunoassays

The hippocampus was homogenized manually using a small pestle in lysis buffer (20 mM Tris–HCl pH 8.0, 10% glycerol, 137 mM NaCl, 0.5 mM sodium vanadate, 1% NP-40, proteases inhibitor cocktail (Sigma-Aldrich Corp, MI, USA) and 1 mM polymethane sulfonyl floride (PMSF). Homogenates were centrifuged at 14,000×g for 5 min, 4 °C. Using a commercially available kit, the total protein concentrations were estimated (BioRAD, Hercules, CA, USA). To quantify GSH, 5% 5-Sulfosalicylic Acid (SSA) was added to the homogenate. Then the samples were assayed for total GSH/GSSG according to manufacture instructions (Glutathione Assay Kit, Sigma-Aldrich Corp, MI, USA).

Glutathione peroxidase (GPx) was assayed using a commercially available kit as per kit’s manufacturer’s instructions (Glutathione Peroxidase Cellular Activity Assay Kit, Sigma-Aldrich, MI, USA). Similarly, Catalase and SOD activities were measured using a commercially available kits (Cayman Chemical, Ann Arbor, MI, USA). Plates were read at kit’s specified wave lengths using an automated reader (Epoch Microplate Spectrophotometer, Bio-tek instruments, Highland Park, Winooski, USA).

Homocysteine level in serum was measured by using rat homocysteine Kit from Cusabio Biotech Co (CSB-E13376r, USA). Briefly, 100 μl Biotin-antibody working solution was added to 100 μl tissue homogenates. The mixture was incubated for 60 min at 37 °C, and then it was washed. Thereafter, 100 μl of HRP-avidin working solution was added and incubated for 60 min at 37 °C. After that, 90 μl of TMB substrate was added and it was incubated for 30 min at 37 °C. Finally, 50 μl of stop solution was added and the plates were read at 450 nm using the automated reader.

Statistical Analysis

The statistical analysis was done using Graph Pad Prism (4.0) for Windows. The numbers of errors made by animals were compared using two-way AVOVA; succeeded by Bonferroni posttest. Repeated measures factor was time and between-subjects factor was the groups, and they were treated as independent variables. Oxidative stress biomarkers were compared using one-way AVOVA; succeeded by Bonferroni posttest. Significance level was set at P < 0.05. The mean ± SEM was used to represent all values.

Results

l-Methionine Treatment Increases Serum Homocysteine Levels

l-Methionine treatment induced significant increase in the levels of serum homocysteine. On the other hand, treatment with caffeine did not alter serum homocysteine levels (Fig. 1).

Fig. 1
figure 1

Levels of homocysteine in serum. Comparison of control (Control), Caffeine (CAF), l-methionine (Meth), l-methionine with Caffeine (Meth+CAF). l-methionine supplementation resulted in significant increase in serum methionine levels in the Meth and Meth+CAF groups. Each point is the mean ± SEM. *Significant difference from other groups, (P < 0.05, n = 12)

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Caffeine Prevents l-Methionine-Induced Memory Impairment

A marked reduction of errors in the learning phase (trials 1–12; Fig. 2) was detected in all animal groups, suggesting learning the RAWM task. The learning performance was similar among all animal groups. This suggests neither l-methionine nor caffeine affected learning performance in the RAWM. By trial 12 of the learning phase, all groups showed similar number of errors indicating that animals have learned to the same extent (Fig. 2).

Fig. 2
figure 2

Animals’ performance during the learning phase of the RAWM. Comparison of control (Control), Caffeine (CAF), l-methionine (Meth), l-methionine with Caffeine (Meth with CAF). Each animal was trained for six consecutive trials separated by 5-min rest, then another six consecutive trials (the learning phase). No difference was observed in learning performance among experimental groups. Values are mean ± SEM. n = 12/group

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In short-term memory test at 30 min, the end of the last learning trial, animals in the Control, Caf, and l-Meth+Caf groups committed similar number of errors in finding the hidden platform in the RAWM. In contrast, the l-Meth group committed significantly more errors to find the hidden platform than other experimental groups (Fig. 3a). Thus, caffeine prevented impairment of short-term memory in l-Meth group as showed by an absence of significant difference from the control group.

Fig. 3
figure 3

Caffeine prevents short- and long- term memory impairment induced by l-methionine supplementation. Short-term memory test was performed at 30 min (a), and long-term memory tests 5 h (b) and 24 h (c) after the last trial of the learning phase. Each point is the average values ± SEM from 12 rats. *Significant difference from other groups, (P < 0.05)

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In long-term memory tests, which were done 5 h and 24 h after the end of the last learning trial, the l-Meth group made significantly more errors to find the hidden platform than the other experimental groups. Whereas, caffeine treatment prevented the long-term memory impairment in L-Meth group as showed by lack of significant difference from the control group (Fig. 3b, c). This result indicates that chronic caffeine treatment prevented chronic l-methionine administration induced long-term memory impairment in the RAWM paradigm.

The Effect of l-Methionine and/or Caffeine on Hippocampus Oxidative Stress Biomarkers

No change was observed among experimental groups in the level of GSH (Fig. 4a). The levels of GSSG were significantly increased in association with l-methionine. This increase was prevented by the treatment with caffeine (Fig. 4b). Concerning the ratio GSH/GSSG, it was markedly reduced by l-methionine treatment, which was prevented by treatment of caffeine (Fig. 4c). L-methionine treatment is associated with a significant decrease in hippocampus GPx activity. On the other hand, treatment with caffeine prevented l-methionine induced reduction in GPx activity (Fig. 5a). The activity of catalase was significantly reduced by l-methionine treatment. On the other hand, treatment with caffeine normalized l-methionine induced reduction in catalase activity (Fig. 5b). Finally, there was no change observed in the activity of SOD measured as SOD inhibition rate among the studied groups (Fig. 5c).

Fig. 4
figure 4

a No change was observed in GSH levels among experimental groups. b l-methionine increased GSSG levels, and (c) reduced the ratio GSH/GSSG in Meth group compared to other groups. Administration of caffeine prevented these alterations in GSSG and GSH/GSSG ratio induced by l-methionine. Each point is the mean ± SEM. *Significant difference from other groups, (P < 0.05, n = 12)

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Fig. 5
figure 5

l-Methionine reduced activity of a GPx and b catalase in the hippocampus. Comparison of control (Control), Caffeine (CAF), l-methionine (Meth), l-methionine with Caffeine (Meth+CAF). c No change in SOD enzymatic activity in the hippocampus among experimental groups. Each point is the mean ± SEM. *Significant difference from other groups, (P < 0.05, n = 12)

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Discussion

The major finding in the current study is that caffeine treatment prevented impairment of short- and long-term memory induced by chronic l-methionine administration in the RAWM spatial learning and memory tasks. The hippocampus levels of oxidative stress biomarkers, including GSH/GSSG ratio and enzymatic activities of GPx and catalase, which are important for cognitive function, were reduced by chronic l-methionine administration. These effects were prevented by caffeine treatment, indicating the involvement of oxidative stress mechanisms in the interactive effects of l-methionine and caffeine.

Several studies on animals revealed that chronic administration of l-methionine resulted in increasing levels of homocysteine in the blood (Baydas et al. 2005, 2006; Goyal et al. 2010; Koz et al. 2010). This is in agreement with the present results where chronic administration of l-methionine resulted in hyperhomocysteinemia. Homocysteine plays a vital role in oxidative stress, brain defect, cognitive, and memory dysfunction (Baydas et al. 2008; Cito et al. 2010; Ho et al. 2001; Kuszczyk et al. 2009; Lafon-Cazal et al. 1993; McVeigh and Passmore 2006). Studies have shown that increasing levels of homocysteine increases the risk of dysfunction in endothelial cells that may interfere with blood supply to the brain. This increases the risk of vascular disease and alters reactive oxygen species playing roles in the stimulation of oxidative stress involved in the atherosclerotic process (Majithiya et al. 2005). The link between memory impairment and hyperhomocysteinemia has been demonstrated in the present study and in previous studies (Alzoubi et al. 2014; Bhargava et al. 2018). For example, chronic l-methionine administration has been reported to induce vascular dementia (El-Dessouki et al. 2017; Mangat et al. 2014), and caffeine treatment has been associated with many positive effects, such as improvements of cognitive functioning and prevention of memory impairment (Costenla et al. 2010; Duarte et al. 2012; Ribeiro and Sebastiao 2010).

Besides, the finding of the current study and previous studies was chronic administration of l-methionine results in hyperhomocysteinemia that leads to increase of oxidative stress in the hippocampus and cerebral cortex of adult rats (Alzoubi et al. 2014; Cheng et al. 2016; Kolling et al. 2017; Zeng et al. 2018). Several studies have shown that chronic hyperhomocysteinemia induced oxidative stress in various tissues of animals and human (Makhro et al. 2008; Mujumdar et al. 2001; Wang et al. 2004) and decreased antioxidant protection systems in tissues such as the hippocampus of rats (Baydas et al. 2003, 2006; Tang et al. 2011; Weiss 2005). The beneficial effects of enhancing memory by caffeine via their antioxidant effect have been widely reported (Abreu et al. 2011; Gomez-Ruiz et al. 2007; Lee 2000; Shi et al. 1991; Varma et al. 2010). In the current study, chronic administration of L-methionine induced oxidative stress, through decreasing antioxidant protection systems in the hippocampus, namely, GSH/GSSG ratio, catalase, and GPx, that could lead to short- and long-term memory impairments.

Current results showed that there is no significant variation in the level of GSH among studied groups when compared with control. On the other hand, there is a significant elevation in the level of GSSG and significant decrease in the ratio of GSH/GSSG in l-methionine group when compared with other groups, which means high level of oxidative stress in that condition. This significant variation supports the view of oxidative stress as a precipitating factor for l-methionine induced memory impairment (Maiti et al. 2008). In support of that, current results showed that chronic L-methionine ingestion was associated with significant decrease in hippocampus GPx and catalase enzymatic activities. On the other hand, treatment with caffeine prevented these decreases. Previous studies revealed a decrease in the activity of GPx in rats that were fed with methionine (Baydas et al. 2005) and in stress animal models (Noschang et al. 2009; Upchurch et al. 1997). Preventing decrease in oxidative capacity enzymes such as GPx and catalase could be one possible mechanism by which caffeine can prevent oxidative stress in l-methionine-treated groups.

Interestingly, methionine can exacerbate psychotic episodes in schizophrenia patients (Cohen et al. 1974). Additionally, schizophrenia patients were reported to have higher homocysteine levels, a metabolite of methionine, compared with the general populations (Dietrich-Muszalska et al. 2012; Muntjewerff et al. 2006). Schizophrenia patients also consume more coffee compared with the normal population (Strassnig et al. 2006). It was also suggested that caffeine may have some positive effects on cognition in subjects with schizophrenia (Nunez et al. 2015). Thus, it may be that such behavior of excessive coffee consumption in schizophrenia patients is some kind of self-therapy. Similar line of behavior could extrapolated other dementias related to elevated methionine levels.

In conclusion, caffeine prevents the short- and long-term memory impairments induced by chronic l-methionine administration; possibly thorough preventing reduction in antioxidant protection mechanisms including the GSH/GSSG ratio, catalase, and GPx, during chronic l-methionine administration.