Caffeine citrate effects on gastrointestinal permeability,bacterial translocation and biochemical parameters in newborn rats after long-term oral administration

Abstract

BACKGROUND:

Caffeine is a potent central and respiratory acting agent used in neonatology to treat apnea in premature newborns.

OBJECTIVE:

This study investigates the effects of caffeine orally administered to newborn rats on gastrointestinal permeability, bacterial translocation and different biochemical parameters.

METHODS:

Newborn rats were divided into different groups (N = 06). The treated newborn rats were orally administered with standard caffeine doses (12 mg/kg per day), and the control groups received a placebo. The animals were weighed daily until sacrifice. Blood samples, mesenteric lymph nodes (MLN) and organs were aseptically collected. Furthermore, different biochemical (D-Lactate) and oxidative stress biomarkers (MDA, CAT, SOD and GSH) were examined. Microbiological analyses were performed to assess microbiota alterations and bacterial translocation.

RESULTS:

Preliminary results showed that caffeine administration decreased the level of bacterial translocation over time. The treatment reduced plasma D-lactate levels (p < 0.05). Additionally, caffeine induced a disturbance in the concentrations of biochemical parameters and oxidative stress biomarkers. Indeed, liver enzymes (AST and ALT) were significantly (p < 0.05) risen after caffeine treatment. Glutathione (GSH) levels were significantly higher in caffeine treated groups (75.12±0.32; 51.98±1.12 U/mg; p < 0.05) comparing to control ones (40.82±0.25; 42.91±0.27 U/mg; p < 0.05) in the ileum and the colon, respectively.

CONCLUSIONS:

Thus, besides improving gastrointestinal permeability, our data show that caffeine has beneficial effects on the intestinal antioxidant system.

Keywords

Caffeine neonatal life intestinal permeability bacterial translocation oxidative stress parameters

1 Introduction

The gastrointestinal tract, besides its obvious functions such as digestion and absorption of nutrients into the body, constitutes an effective mucosal barrier and a complex immune organ [1]; protecting the host against the passage of potentially harmful macromolecules and translocation of pathogenic microorganisms. A large population of microorganisms, called microbiota, colonize the gut. The commensal colonization promotes the maturation of mucosal barrier and intestinal immune system, both of which are necessary for maintaining homeostasis [2]. Although the gut acts as a barrier between these microorganisms and the host, gastrointestinal bacteria might translocate under several circumstances, including increased intestinal barrier permeability, deficiencies in the host immune system, and overgrowth of intestinal bacteria [3].

During fetal and neonatal periods, macromolecules are highly transferred through the immature intestinal epithelium. The significant endocytic potential of immature (fetal-type) enterocytes causes increased intestinal permeability at these periods. These cells internalize macromolecules in the luminal content by fluid-phase or receptor-mediated endocytosis. Macromolecules are either digested in digestive vacuoles or transported to the basolateral side of the cell by vesicles [1]. In fetuses and neonates, this property is mainly lost as development and maturation advance, until macromolecular transepithelial transport stops at the moment of so-called “gut closure”.

Caffeine (1,3,7-trimethylxanthine) is a liposoluble drug with 100% bioavailability that is absorbed rapidly and efficiently through the gastrointestinal system [4, 5]. It’s a naturally occurring alkaloid found in the leaves, seeds, and fruits of a variety of plants [6]. Caffeine can be synthesized artificially and used in dietary supplements and medications with different forms of administration [7]. This substance has a variety of physiological effects on different organisms at micromolar levels with a wide therapeutic window [8]. Caffeine is used to treat and prevent apnea of prematurity, a common complication in neonates with a gestational age of 28–33 weeks that is observed in the region of 90% of premature infants weighing less than 1 kg and in 25% of premature infants weighing less than 2.5 kg [9]. The first treatment for apnea of premature newborns was the theophylline used by Kuzenko and Poala [10]. In 1977, it was demonstrated that caffeine is highly effective than theophylline in the stimulation of the central nervous and respiratory systems [11] and has fewer adverse effects [12]. Currently, caffeine citrate represents the drug of choice for premature apnea treatment and prevention of bronchopulmonary dysplasia and respiratory distress syndrome [13].

Several studies have evaluated both short and long-term effects of caffeine in neonates, and primarily described neurodevelopment and cardiovascular outcomes. However, the caffeine effect on newborn gastrointestinal functions and gut microbiota is not clear [14]. This might be because caffeine is absorbed and degraded quickly and completely in the upper gastrointestinal system [15]. Additionally, animal studies have shown that caffeine can cause microbiota dysbiosis, which is likely caused by its antimicrobial properties [16, 17]. Thus, we hypothesized that caffeine, may affect gastrointestinal homeostasis at an early age.

The objective of our study was to investigate the effects of caffeine orally administered to newborn rats on gastrointestinal permeability, bacterial translocation and different biochemical parameters.

2 Material and methods

2.1 Animals

Wistar rats of the opposite sex were used during this study; animals were purchased from the Animal House (Mascara, Algeria). They were housed three to a cage under well hygienic conditions. Temperature and light were controlled (23±2 °C; 12h light/12h dark cycles). Animals were fed ad libitum. The experimental protocol was designed to use the minimum number of animals (N = 06) as well as to keep them from suffering.

2.2 Ethical considerations

The animal experiments were conducted following the guidelines of the Organization for Economic Cooperation and Development (OECD) (protocol n°408, 25 June 2018), and approved by the Ethics committee for animal research of Mustapha Stambouli University (N/Réf: 04/CSF/SNV/2016, 08 November 2016).

2.3 Study design

Females were caged with males for 10 days and after 14 days gestation was detected by abdominal palpation. Neonate’s birth happened after 22 days of mating. On the first day of life (day 1), three series of newborn rats were formed (S1 = 15 days, S2 = 30 days and S3 = 60 days). Each series was divided into 2 groups (N = 06), the first group was treated orally with standard caffeine citrate dose of 12 mg/kg/day (as caffeine base) (CITRATE DE CAFEINE COOPER, Renaudin Laboratory- France), control groups received saline placebo. The animals were weighed daily until the day of sacrifice. A laparotomy was performed; blood and organs samples were aseptically collected from each rat.

2.4 Microbiota assessment

Colon and ileum samples (1g) were aseptically dissected, homogenized and diluted serially to make 10^–3, 10^–5 and 10^–7 concentrations. 0.1 mL of each dilution was plated on the appropriate medium plates (Columbia agar, for total aerobes; Eosin Methylene agar, for Enterobacteria; Schaedler agar, for strict anaerobes and MRS agar for Lactobacillus). Aerobic and anaerobic cultures were incubated at 37°C for 24 h and 72 h, respectively. The colonies counts on the agar plates were represented as colony-forming units per gram of tissue.

2.5 Assessment of bacterial translocation

The existence of a positive culture from the mesenteric lymph nodes (MLNs) and external organs was considered as bacterial translocation [18]. To evaluate the degree of bacterial translocation, MLNs (from the ileum), liver, spleen and thymus specimens were removed aseptically. The tissue samples were rinsed in sterile saline solution to remove any blood. For bacteriological cultures, 0.1 g of each sample was immediately homogenized in 0.9 mL of sterile phosphate-buffered saline (PBS). 0.1 mL aliquot of each sample was cultured on Columbia agar, Eosin Methylene Blue agar (EMB agar) and Schaedler agar for total aerobic bacteria, Enterobacteria and strictly anaerobic bacteria detection, respectively. The cultures were incubated at 37°C for 48 hours. Anaerobic jars were used for anaerobic cultures [19]. Bacterial translocation was considered positive when the count was higher than 100 colonies forming units/g of tissue [20].

2.6 Analysis of intestinal permeability

The d-lactic acid level was measured to assess the changes in intestinal permeability. Using a spectrophotometric assay [21], plasma samples, ileum and colon homogenates were deproteinized and neutralized with perchloric acid and potassium hydroxide, respectively. D-lactate level was then quantified using D-lactate dehydrogenase and alanine aminotransferase in a series of linked enzymatic processes.

2.7 Serum biochemical parameters assays

After each period of the experiment, newborn rats were sacrificed by Chloroform inhalation. Blood was collected through the aorta vein from control and caffeine-treated rats. Blood was centrifuged at 1500g for 15 min to collect the serum.

Serum total cholesterol(TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), the concentrations of serum triacylglycerol (TG), aspartate transaminase (AST), alanine transaminase (ALT), urea, creatinine (CREA), bilirubin (BL), Lactate dehydrogenase (LDH) and phosphatase alkaline (PAL) levels were determined using the Respons 920- DiaSys Diagnostic Systems GmbH analyzer (65558 Holzheim, Germany).

2.8 Measurement of malondialdehyde (MDA) content, catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) activities in the ileum the and colon

The ileum and colon tissues were thawed and homogenized on ice in the ratio of 2 g tissue for 8 mL of phosphate buffer saline. The homogenates were then centrifuged for 15 min at 10000 rpm at 4°C and supernatants were used for the different analyses.

The MDA content was assessed by the thiobarbiturate reaction using the method of Chiva and et al. [3]. The enzymatic activity of SOD (superoxide dismutase) was evaluated utilizing the spectrophotometric method developed by Marklund and Marklund [22]. Catalase was assessed calorimetrically (at 240 nm) as described by Caliborne [23]. Glutathione (GSH) was assessed as per Moron et al. [24]. The total protein level was measured at 540 nm using the Biuret reagent and bovine serum albumin as the standard, as reported by Gornall et al. [25].

2.9 Statistical analysis

Data of the present study were expressed as means±standard deviation (SD). To compare between the treated and control groups, a statistical analysis was done using the Student’s t-test. A p value < 0.05 was considered significant.

3 Results

3.1 Effect of caffeine on body weight

The main results of body weight are illustrated in Fig. 1. The body weight increased in both treated and non-treated groups in a time-dependent manner. Animals receiving caffeine treatment did not differ in weight from controls. However, a significant (p < 0.05) decrease in body weight was noticed in the last treated group (60 days of treatment).

Fig. 1

Effect of caffeine administration on rats weight (g) (n = 06). CT: control group; CF: caffeine treated group; ^*Significant difference between CT and CF (p < 0.05).

3.2 Effect of caffeine on the microbiota

As shown in Fig. 2, and compared to the control groups, the ileum and colon colonies counts of total aerobes and strict anaerobes exhibited no changes after caffeine administration. However, the counts of Enterobacteria and Lactobacillus were slightly lower in treated newborns compared to controls but the differences were not significant.

Fig. 2

Effect of caffeine administration on microbial counts in the ileum and colon. CT: control group; CF: caffeine treated group; TAB: total aerobic bacteria; TANB: total anaerobic bacteria; ETR: Enterobacteria; LAB: Lactobacillus. 15D, 30D, 60D: days of treatment. CFU/g: colony-forming unit per gram of tissue.

3.3 Effect of caffeine on bacterial translocation

Our data (Table 1) revealed that bacteria were observed in mesenteric lymph nodes (MLNs), Liver and spleen of the 15 days control rats. However, we noticed that BT had decreased in the treated groups. Treatment with caffeine reduced BT in MLNs and distant organs. Moreover, a total absence of translocation was observed in caffeine-treated group at 30 days of life compared to controls. No BT was pronounced in the 60 days treated and untreated groups.

Table 1
Bacterial translocation to MLNs, liver, spleen and thymus in treated and control groups

Groups MLNs Liver Spleen Thymus

15 D CT 4/6 2/6 2/6 0/6

CF 2/6 1/6 0/6 0/6

30 D CT 2/6 2/6 1/6 0/6

CF 1/6 0/6 0/6 0/6

60 D CT 0/6 0/6 0/6 0/6

CF 0/6 0/6 0/6 0/6

Groups	MLNs	Liver	Spleen	Thymus
15 D	CT	4/6	2/6	2/6	0/6
	CF	2/6	1/6	0/6	0/6
30 D	CT	2/6	2/6	1/6	0/6
	CF	1/6	0/6	0/6	0/6
60 D	CT	0/6	0/6	0/6	0/6
	CF	0/6	0/6	0/6	0/6

CT: control group, CF: caffeine treated group. Data are given as (d/e) = positive culture/total rat of group.

3.4 Effect of caffeine on intestinal permeability

Figures 3 and 4 show the mean values of D-lactate levels in control and caffeine-treated groups. Our data revealed that treatment with caffeine at an early age decreased plasma D-lactate concentrations compared to controls. Actually, levels of D-lactate were significantly lowered from 1.76±0.19 to 0.61±0.20μg/ml in the blood serum of the 15 days old treated rats (p < 0.05). Likewise, reduction in D-lactate levels was also observed in ileum and colon homogenates of 15 days and 30 days old treated pups. However, no significant changes in the 60 days treated groups were noticed.

Fig. 3

D-lactate levels in the ileum and colon homogenates of control and caffeine treated newborn rats (mean±SD) (CT: control group; CF: caffeine treated group). ^* Significant differences between CT and CF (p < 0.005).

Fig. 4

D-lactate levels in the blood serum of control and caffeine treated newborn rats (mean±SD) (CT: control group; CF: caffeine treated group). ^* Significant differences between CT and CF (p < 0.05).

3.5 Effect of caffeine on serum biochemical parameters

Mean biochemical parameters concentrations of treated and non-treated newborns are summarized in Table 2. We observed that administration of caffeine exhibit changes on most of the serum parameters levels in comparison to control groups. Triglycerides levels were considerably lowered in the 60 days treated rats when compared with controls (from 0.94±0.10 to 0.57±0.22 g/l). Likewise, a significant reduction in total cholesterol level was obtained in 30 days treated group. In fact, total cholesterol level declined from 0.57±0.02 to 0.53±0.02 g/l. In addition, low-density lipoprotein cholesterol (LDL) levels did not differ statistically between the treated and untreated groups (Table 2). Although treated rats had lower levels of high-density lipoprotein cholesterol (HDL) than controls, the difference was not statistically significant. Compared to controls, caffeine raised significantly (p < 0.05) liver enzymes (AST and ALT) in the 60 days treated group. While AST and ALT levels remained unchanged in the other groups. Caffeine also reduced urea and creatinine levels in all treated groups as compared to controls. Moreover, administration of caffeine resulted in 301±48.30, 251±32.02 and 281±71.21 reduction of alkaline phosphatase (ALP) levels after 15, 30 and 60 days, respectively. Compared to controls, lactate dehydrogenase (LDH) levels enhanced significantly after 15 days of treatment. Furthermore, a significant decrease (p < 0.05) in the level of total bilirubin was observed in 15 days and 30 days treated groups. Additionally, caffeine altered direct bilirubin levels in the 30 days treated group.

Table 2
Caffeine effects on serum biochemical parameters levels (n = 6)

Parameters and groups 15D 30D 60D

CT CF CT CF CT CF

AST (U/l) 148.60±6.08 143.33±2.16 162±5.15 184±34.2 165±6.30 198.5±24.80

p value 0.087 0.177 0.019^*

ALT (U/l) 53.33±3.26 61.83±18.17 49±11.66 79.5±36.76 60.5±2.16 107.16±42.10

p value 0.307 0.100 0.042^*

ALP (U/l) 493±52.30 301±48.30 355.66±37.47 251±32.02 289±38.75 281±71.21

p value 0.00006^* 0.0004^* 0.811

LDH (U/l) 1316±5.83 1349.33±7.89 1134.66±265.71 880.66±30.45 783.83±236.41 982.16±68.57

p value 0.00001^* 0.066 0.097

TG (g/l) 0.39±0.05 0.35±0.07 0,67±0.07 0.65±0.09 0.94±0.10 0.57±0.22

p value 0.358 0.751 0.007^*

TC (g/l) 0.42±0.03 0.451±0.03 0.57±0.02 0.53±0.02 0.61±0.12 0.51±0.08

p value 0.225 0.017^* 0.107

HDL (g/l) 87.83±4.53 83.16±8.47 93.16±7.30 81±6.92 107.33±15.50 84.83±10.45

p value 0.269 0.831 0.161

LDL (g/l) 0.84±0.05 0.73±0.14 0.87±0.36 0.93±0.10 0.85±0.50 0.91±0.20

p value 0.137 0.728 0.804

Urea (g/l) 0.21±0.02 0.15±0.01 0.35±0.02 0.29±0.02 0.34±0.06 0.30±0.06

p value 0.001^* 0.0002^* 0.276

Creatinine (mg/l) 5.18±0.16 5.55±0.28 6.56±0.17 6.13±0.24 7.41±0.14 6.61±0.35

p value 0.024^* 0.006^* 0.001^*

TB (mg/ml) 10.46±0.45 9.71±0.54 9.40±0.34 7.78±0.42 7.48±0.78 7.26±0.94

p value 0.024 0.00003^* 0.673

DB (mg/ml) 2.26±0.07 1.95±0.49 2.35±0.16 1.54±0.27 1.73±0.37 1.62±0.14

p value 0.189 0.0002^* 0.507

Parameters and groups	15D	30D	60D
AST (U/l)	148.60±6.08	143.33±2.16	162±5.15	184±34.2	165±6.30	198.5±24.80
p value	0.087	0.177	0.019^*
ALT (U/l)	53.33±3.26	61.83±18.17	49±11.66	79.5±36.76	60.5±2.16	107.16±42.10
p value	0.307	0.100	0.042^*
ALP (U/l)	493±52.30	301±48.30	355.66±37.47	251±32.02	289±38.75	281±71.21
p value	0.00006^*	0.0004^*	0.811
LDH (U/l)	1316±5.83	1349.33±7.89	1134.66±265.71	880.66±30.45	783.83±236.41	982.16±68.57
p value	0.00001^*	0.066	0.097
TG (g/l)	0.39±0.05	0.35±0.07	0,67±0.07	0.65±0.09	0.94±0.10	0.57±0.22
p value	0.358	0.751	0.007^*
TC (g/l)	0.42±0.03	0.451±0.03	0.57±0.02	0.53±0.02	0.61±0.12	0.51±0.08
p value	0.225	0.017^*	0.107
HDL (g/l)	87.83±4.53	83.16±8.47	93.16±7.30	81±6.92	107.33±15.50	84.83±10.45
p value	0.269	0.831	0.161
LDL (g/l)	0.84±0.05	0.73±0.14	0.87±0.36	0.93±0.10	0.85±0.50	0.91±0.20
p value	0.137	0.728	0.804
Urea (g/l)	0.21±0.02	0.15±0.01	0.35±0.02	0.29±0.02	0.34±0.06	0.30±0.06
p value	0.001^*	0.0002^*	0.276
Creatinine (mg/l)	5.18±0.16	5.55±0.28	6.56±0.17	6.13±0.24	7.41±0.14	6.61±0.35
p value	0.024^*	0.006^*	0.001^*
TB (mg/ml)	10.46±0.45	9.71±0.54	9.40±0.34	7.78±0.42	7.48±0.78	7.26±0.94
p value	0.024	0.00003^*	0.673
DB (mg/ml)	2.26±0.07	1.95±0.49	2.35±0.16	1.54±0.27	1.73±0.37	1.62±0.14
p value	0.189	0.0002^*	0.507

CT: control group, CF: caffeine treated group, AST: Aspartate Transaminase, ALT: Alanine Transaminase, LDH: Lactate Dehydrogenase, ALP: Alkaline Phosphatase, TG: Triglycerides, TC: Total Cholesterol, HDL: high-density lipoprotein cholesterol, LDL: low-density lipoprotein cholesterol, TB: total bilirubin, DB: direct bilirubin. All values are expressed as mean±SD (n = 6). ^*Significant differences between CT and CF (p < 0.05).

3.6 Effect of caffeine on CAT, SOD, GSH activities and MDA content in the ileum and the colon

Caffeine citrate’s effect on the gut antioxidant system was also investigated. Levels of oxidative stress biomarkers measured in the ileum and the colon homogenates are presented in Figs. 5 , 7 and 8. The results showed that treatment with caffeine slightly decreased Malondialdehyde (MDA) levels (Fig. 5), increased Catalase (CAT) and Superoxide Dismutase (SOD) levels in treated groups as compared to control groups in both intestine fragments. Indeed, catalase enhancement was statistically significant in the ileum after 60 days of treatment. Moreover, SOD levels were statistically significant in the ileum (15 days) and the colon (15 and 30 days).

Fig. 5

Caffeine Effect on the content of MDA in the ileum and colon homogenates (CT: control group; CF: caffeine treated group). ^* Significant differences between CT and CF (p < 0.05).

Fig. 6

Caffeine effect on the activities of CAT enzyme in the ileum (CT: control group; CF: caffeine treated group). ^* Significant differences between CT and CF (p < 0.05).

Fig. 7

Caffeine effect on the activities of SOD enzyme in the colon (CT: control group; CF: caffeine treated group). ^* (p < 0.01) ^** (p < 0.0005) Significant differences between CT and CF.

Fig. 8

Caffeine effect on the activity GSH in the ileum and colon (CT: control group; CF: caffeine treated group). ^* (p < 0.05) ^** (p < 0.005) Significant differences between CT and CF.

Glutathione (GSH) levels were significantly higher (Fig. 8) in caffeine treated groups (75.12±0.32; 51.98±1.12 U/mg; p < 0.05) comparing to control ones (40.82±0.25; 42.91±0.27 U/mg; p < 0.05) in the ileum and the colon, respectively, at 15, 30 and 60 days of treatment.

4 Discussion

Caffeine has raised many health concerns over the past decade. Many reports have investigated the various impacts of caffeine. It is a stimulant for the central nervous system. After oral intake, caffeine plasma level reached its maximum after 30–120 min. Caffeine may cross all biological membranes due to its hydrophobic characteristics [26]. Furthermore, it is an adenosine-receptor antagonist [27], and can affect all tissue expressing adenosine receptors. The current study was designed to determine the effects of caffeine administration on gastrointestinal permeability, bacterial translocation (BT) and different biochemical and oxidative stress parameters in newborn rats.

Changes in the weight of rodents after exposure to caffeine have been reported in several studies. Our results showed that body weight increase was not significantly suppressed in neonate rats after 15 and 30 days (Fig. 1) of treatment. This finding corroborate the results of Kovacs et al. [28], who reported no significant difference in body weight between subjects receiving caffeine with green tea and those ingesting only placebo after 13 weeks with a very-low energy diet. Moreover, Schmitt et al. [29] did not report any significant changes in weight during the 28-day exposure to p-synephrine, ephedrine, salicin and caffeine mixture used as an anti-obesity. However, a previous study demonstrated that treatment with caffeine citrate appears to cause temporary suppression of body weight gain in newborn rats. The difference in weight loss between treated and control groups rose and became highly significant on the 30th day of life [17]. Furthermore, many researchers have shown that long-term intake of caffeine lowered body weight in rodents [30 –32]. Those results are in agreement with our findings showing a significant decrease in weight after 60 days of treatment.

The integrity of intestinal barriers, which include mechanical, chemical, biological, and immunological barriers, determines intestinal permeability. When a barrier is breached, the gut’s permeability increases. The microbiota is a component of the biological barrier, which has both positive and negative impacts on intestinal permeability [33]. Anaerobes (Firmicutes and Bacteroidetes), are the main bacteria found in the gastrointestinal tract and account for around 90% of all microbiota [34]. No differences among anaerobic counts between caffeine-treated rats and controls were observed in our study (Fig. 2). In the same line, Kleber Silveira et al. [16] found no significant changes in anaerobic bacteria abundance in caffeine-treated groups when compared to the Guarana-treated and control groups. Our data showed that caffeine treatment decreased the number of intestinal Enterobacteria and Lactobacillus in the treated groups compared to controls. This decrease may be related to the antibacterial properties of caffeine [35]. In a previous study, our team showed that caffeine treatment during postnatal period in newborn rats, did not drastically alter the kinetics of establishment of the intestinal microbiota, only Enterobacteria were significantly lower in different intestinal segments of the treated groups (p < 0.05) [17]. Moreover, caffeine may affect negatively the microbiota and reduce the levels of Lactobacillus, according to Kleber Silveira et al. [16].

The mechanism by which caffeine altered gut aerobic and anaerobic bacterium concentrations was not explored in this investigation. More research on the impact of caffeine on the microbiota communities in the gut is required.

The translocation of bacteria (BT) from the intestinal lumen to the blood stream and systemic organs is due to damages in the intestinal barrier, alteration in the normal flora or the immaturity of the gastrointestinal tract. Indeed, the gut immune system’s immaturity enhance the danger of systemic infections [36]. Bacteria with a high potential for pathogenicity have can cross through the gut barrier [37]. In the current study, we showed that bacterial translocation occurred at an early age, representing a natural phenomenon in neonates. Actually, we observed the presence of bacteria (especially Enterobacteria) in MLNs and distant organs such as the liver and spleen of newborn rats after 15 days of life. Supporting our results, Berg R.D. [38] showed that viable bacteria detected in the MLNs indicate bacterial translocation from the gut lumen. Wiest et al. [39] revealed that Gram-negative aerobes translocate easily than anaerobes across the intestinal epithelium. However, the incidence of BT was lower in caffeine-treated rats. Coffee components such as caffeine, caffeic acid, and trigonelline, according to Almeida et al. [35] have antibacterial effects against pathogenic microorganisms. Indeed, coffee inhibited the growth of Enterobacteria the most commonly bacteria found in the gut [35]. The decrease in BT is explained, at least in part, by alterations in intestinal microbiota found following caffeine administration in the current investigation.

We hypothesized that D-lactate assessment could confirm the bacterial translocation phenomena in newborn rats during the treatment with caffeine. According to our results, D-lactate decreased remarkably during exposure to caffeine. In fact, in the 15 days old treated neonates, levels D-lactate in plasma, ileum and colon homogenates decreased significantly (p < 0.05) (1.76±0.19, 113±0.85 and 143±5.01 to 0.61±0.20, 89±1.96 and 96±1.65μg/ml, respectively). D (-)-lactate is synthesized by numerous species of anaerobic bacteria present in the gastrointestinal system [40]. The elevation of D-lactate concentration in the blood is commonly caused by bacterial over growth and elevated intestinal permeability, thereby allowing D-lactate to enter the systemic circulation [41, 42]. Indeed, earlier studies have reported a significant increase in blood D-lactate levels as consequence of bacterial colonization, systemic infections, or gastrointestinal disorders [43].

Taken together, those results confirms the hypothesis that treatment with caffeine accelerates intestinal maturation and controls bacterial translocation in newborn rats.

In the present study, we determined different biochemical parameters levels in plasma of treated and control newborn rats using the Respons 920- DiaSys Diagnostic Systems GmbH analyzer (65558 Holzheim, Germany). The considerable reduction in serum lipid profile is consistent with a precedent study reporting that caffeine treatment decreased triglyceride levels in rats [44]. In fact, our results showed that caffeine treatment suppressed triglycerides levels in the 60 days treated rats (from 0.94±0.10 to 0.57±0.22 g/l). Similarly, a significant reduction in serum total cholesterol levels was obtained in the 30D treated group (from 0.57±0.02 to 0.53±0.02 g/l). In addition, there were no significant changes in HDL-C and LDL-C values between the treated and untreated groups (Table 2).

Aminotransferase levels in the blood are used as a clinical measure of liver health. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two well-known enzymes involved in liver metabolism. Chronic liver disease is frequently detected when ALT and AST levels are increased. Furthermore, several studies demonstrated that ALT and AST are linked to metabolism disorders [45]. Caffeine itself is a bioactive compound related to metabolic features in humans [46]. Our data showed that the enzymes (ALT and AST) rose significantly after 60 days of caffeine administration. Supporting our results and according to Boekschoten et al., coffee bean extracts can raise ALT and AST levels regardless of kahweol concentration [47]. In addition, Urgert et al. discovered that drinking strong coffee regularly might directly enhance the level of circulating ALT [48]. Moreover, Onuegbu et al. found that coffee intake can increase ALT and AST levels depending on gender [49]. In the same line, Handayani et al. showed that administration of ethanol extract of Robusta coffee enhanced the levels of ALT and AST in Wistar albino rats [50]. As a result, the connection between coffee intake and ALT and AST levels may be influenced by the research design. In contrast to epidemiological studies, participants in these randomized controlled trials (RCTs) always drank coffee for a brief period (around only 1 month) [46]. Caffeine administration may cause various acute responses in the liver/body, which might explain the momentarily raised levels of ALT and AST. Dianzani et al. [51] reported that the caffeine peroxidative effect in fatty liver tissue could be the origin of the enzymes leakage. Observations of Manne and Saab [52] support our results. To fully understand the issues at hand, several long-term RCTs are required.

Alkaline phosphatase levels (ALP) were decreased significantly (p < 0.05) in treated rats when compared to the controls. Confirming our results, previous works have found a similar reduction in rats given filtered and unfiltered caffeine [53]. Thus, Nyblom et al. [54] suggested that caffeine or other components of coffee target liver enzymes.

Our results demonstrated that caffeine administration to the newborn rats slightly decreased the levels of urea and creatinine compared to the controls. In all groups, the differences were statistically significant (p < 0.05). Protein catabolism produces urea, whereas creatine metabolism produces creatinine, which is a waste product of muscle. Its presence in the blood is a sign of renal disease [55]. The rise in urea and creatinine levels shows that caffeine intake may affect renal function.

Total and direct bilirubin levels of test groups were reduced (p < 0.05) compared to the controls (30 days). Our results are in line with the observations of Emmanuel et al. [56]. They reported that the intake of caffeine at different doses decreases bilirubin levels.

Oxidative stress is characterized by the disturbance of reactive oxygen species (ROS) levels in the cell nucleus either by elevation or by reduction of ROS, thereby disturbing signaling pathways and causing oxidative changes of cellular components. Cell death can occur as a result of necrosis or apoptosis [57]. Antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) prevent a state of oxidative stress by decomposing ROS [58].

Findings of this investigation showed that caffeine treatment did not generate oxidative damage in lipids of ileum and colon tissues. Indeed, MDA compounds production (Fig. 5) was reduced in treated groups (0.74 nmol/mg of tissue) compared to the control groups (0.98 nmol/mg of tissue). Several studies have shown that caffeine could have a protective role against cellular damage [59, 60]. Nikolic et al. [61] reported that caffeine is considered an antioxidant substance with beneficial effects due to its metabolites, 1-methylxanthine and 1-methyluric acid, which are extremely effective antioxidants. Thus, these studies confirmed that caffeine had antioxidant effects that protect tissue from free radical damage through the reduction or elimination of ROS.

Furthermore, the treatment with caffeine caused no-significant enhancement in CAT activity in ileum and colon tissues (Fig. 6). However, a significant increase of the enzyme activity was observed in the ileum tissue of 60 days treated group (10.12 mM/min/mg) compared to the control group (8.73 mM/min/mg) (p < 0.05). The degradation of intracellular hydrogen peroxide (H₂O₂) requires the existence of CAT. It promotes the breakdown of H₂O₂ into water (H₂O) and oxygen while avoiding the production of free radicals. Moreover, SOD was significantly elevated in the ileum in the 15 days treated group compared to its respective control group. In the colon, SOD levels rose in the 15 and 30 days treated groups. Superoxide radicals are dismutated into hydrogen peroxide and molecular oxygen by the SOD enzyme [62].

Reduced glutathione (GSH) is an important endogenous non-enzymatic antioxidant. The tripeptide GSH act as a cofactor for other enzymes or reacts directly with ROS. In the present work, we observed that caffeine administration enhanced the concentration of glutathione (Fig. 8). In the same line, Renata V.A. et al. demonstrated that caffeine ingestion raised the concentration of GSH in rat brains [63].

Undoubtedly, caffeine has beneficial effects on the antioxidant properties in the gastrointestinal tract. It has been reported that the major molecular target of caffeine, adenosine receptors, are engaged in the control of ROS generation, influencing the origin and effect of free radicals in neuronal and other biological systems [5, 63].

Our study has several limitations. Other intestinal permeability biomarkers could be measured. For example, Diamine oxidase assesses the amount of mucosal injury and hence provides an indirect measure of intestinal permeability. Trans-epithelial electrical resistance for intestinal permeability measure. Additionally, tight junction proteins (TJ) assays to assess intestinal barrier function.

In conclusion, the results of this research revealed a potential association between caffeine administration and gastrointestinal changes in newborn rats. Caffeine improved gastrointestinal permeability through the maturation of the intestinal barrier. Indeed, it was able to decrease bacterial translocation to systemic organs and reduce D-Lactate levels in the blood system. Moreover, caffeine exhibited a protective property against intestinal oxidative stress in neonate rats. Despite the limitations, our preliminary results could open new perspectives in pediatric gastroenterology and support the addition of caffeine to Neonatal Intensive Care Units in our Hospitals. Further studies are needed to confirm caffeine effects at a micro-scale and provide relevant clinical information.

Footnotes

Acknowledgments

The authors would like to acknowledge the Ministry of High Education and Scientific Research of Algeria and Laboratory of Bioconversion, Microbiology Engineering and Health Safety (LBGMSS) of Mustapha Stambouli (Mascara) for supporting this work.

Conflicts of interest

The authors declare no conflict of interest to report.

Funding

The Ministry of High Education and Scientific Research of Algeria and Laboratory of Bioconversion, Microbiology Engineering and Health Safety (LBGMSS) of Mustapha Stambouli (Mascara).

References

Weström

, Arévalo Sureda

, Pierzynowska

, Pierzynowski

, Pérez-Cano

. The immature gut barrier and its importance in establishing immunity in newborn mammals. Frontiers in Immunology. 2020;11:1153.

Hayes

, Dong

, Galipeau

, Jury

, McCarville

, Huang

,... & Verdu

Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis. Scientific Reports. 2018;8(1):1–14.

Chiva

, Guarner

, Peralta

, et al. Intestinal mucosal oxidative damage and bacterial translocation in cirrhotic rats. European Journal of Gastroenterology & Hepatology. 2003;15(2):145–50.

Paluska

. Caffeine and exercise. Current Sports Medicine Reports. 2003;2(4):213–9.

Amer

, et al. Caffeine intake decreases oxidative stress and inflammatory biomarkers in experimental liver diseases induced by thioacetamide: Biochemical and histological study. International Journal of Immunopathology and Pharmacology. 2017;30(1):13–24.

Banerjee

, et al. Fatal caffeine intoxication: a series of eight cases from 1999 to 2009. Journal of Forensic Sciences. 2014;59(3):865–8.

Ruiz-Moreno

, Lara

, Salinero

, de Souza

, Ordovás

, Del Coso

. Time course of tolerance to adverse effects associated with the ingestion of a moderate dose of caffeine. European Journal of Nutrition. 2020:1–10.

Santos-Silva

, et al. Evaluation of caffeine effects on biochemical and genotoxic biomarkers in the neotropical freshwater teleost Prochilodus lineatus. Environmental Toxicology and Pharmacology. 2018;58:237–42.

Rigatto

. Control of ventilation in the newborn. Annual Review of Physiology. 1984;46(1):661–74.

10.

Kuzenko

, Poala

. Apneic attaks in the newborn treated with aminophylline. Arch Dis Child. 1973;48:404–6.

11.

Zulqarnain

, Hussain

, Suleri

, et al. Comparison of Caffeine versus Theophylline for apnea of prematurity. Pakistan Journal of Medical Sciences. 2019;35(1):113.

12.

Nakaoka

, Kawasaki

, Inomata

, Makimoto

, Yoshida

. Caffeine toxicity in a preterm neonate. Pediatrics & Neonatology. 2017;58(4):380–1.

13.

Henderson-Smart

, Steer

. Caffeine versus theophylline for apnea in preterm infants. Cochrane Database of Systematic Reviews. 2010(1).

14.

Schmidt

, Roberts

, Davis

, et al. Long-term effects of caffeine therapy for apnea of prematurity. New England Journal of Medicine. 2007;357(19):1893–902.

15.

Gao

, Xie

, Kong

, Liu

, Sun

, Xiong

, ... & Xiang

. Polyphenol-and caffeine-rich postfermented pu-erh tea improves diet-induced metabolic syndrome by remodeling intestinal homeostasis in mice. Infection and Immunity. 2018;86(1):e00601–17.

16.

Kleber Silveira

, Moresco

, Mautone Gomes

, da Silva Morrone

, Kich Grun

, Pens Gelain

,... & Fonseca Moreira

. Guarana (Paullinia cupana Mart.) alters gut microbiota and modulates redox status, partially via caffeine in Wistar rats. Phytotherapy Research. 2018;32(12):2466–74.

17.

Moumen

, Tir Touil

, Léké

, et al. Establishment of the intestinal microflora and regulation of bacterial translocation after caffeine citrate treatment during postnatal period in rat. Archives de pédiatrie: organe officiel de la Société française de pédiatrie. 2012;19(10):1015–20.

18.

Wang

, Zhang

, Zuo

, et al. Bifidobacteria may be beneficial to intestinal microbiota and reduction of bacterial translocation in mice following ischaemia and reperfusion injury. British Journal of Nutrition. 2013;109(11):1990–8.

19.

Lei

, Bi

, Chen

, et al. Glucagon-like peptide-2 improves intestinal immune function and diminishes bacterial translocation in a mouse model of parenteral nutrition. Nutrition Research. 2018;49:56–66.

20.

, Chen

, Zhang

, et al. Protective effect of glutamine-enriched early enteral nutrition on intestinal mucosal barrier injury after liver transplantation in rats. The American Journal of Surgery. 2010;199(1):35–42.

21.

Szalay

, Umar

, Khadem

, et al. Increased plasma D-lactate is associated with the severity of hemorrhagic/traumatic shock in rats. Shock. 2003;20(3):245–50.

22.

Marklund

, Marklund

. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry. 1974;47(3):469–74.

23.

Caliborne

. Assay of catalase. In: Greenwald RA, editor. Handbook of Oxygen Radical Research. Florida: CRC Press. 1985:196–201.

24.

Moron

, Depierre

, Mannervik

. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et biophysica acta (BBA)-general subjects. 1979;582(1):67–78.

25.

Gornall

, Bardawill

, David

. Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry. 1949;177(2):751–66.

26.

Fredholm

, Bättig

, Holmén

, et al. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacological Reviews. 1999;51(1):83–133.

27.

Van Soeren

, Graham

. Effect of caffeine on metabolism, exercise endurance, and catecholamine responses after withdrawal. Journal of Applied Physiology. 1998;85(4):1493–501.

28.

Kovacs

, Lejeune

, Nijs

, et al. Effects of green tea on weight maintenance after body-weight loss. British Journal of Nutrition. 2004;91(3):431–7.

29.

Schmitt

, Arbo

, Lorensi

, et al. Gender differences in biochemical markers and oxidative stress of rats after 28 days oral exposure to a mixture used for weight loss containing p-synephrine, ephedrine, salicin, and caffeine. Brazilian Journal of Pharmaceutical Sciences. 2016;52(1):59–68.

30.

Zheng

, Sayama

, Okubo

, et al. Anti-obesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. In vivo. 2004;18(1):55–62.

31.

Shimoda

, Seki

, Aitani

. Inhibitory effect of green coffee bean extract on fat accumulation and body weight gain in mice. BMC Complementary and Alternative Medicine. 2006;6(1):9.

32.

Tofovic

, Salah

, Jackson

, et al. Early renal injury induced by caffeine consumption in obese, diabetic ZSF1 rats. Renal Failure. 2007;29(7):891–902.

33.

Cai

, Ran

, Li

, Wang

, Zhou

. Intestinal microbiome and permeability in patients with autoimmune hepatitis. Best Practice & Research Clinical Gastroenterology. 2017;31(6):669–73.

34.

Barandouzi

, Lee

, Maas

, Starkweather

, Cong

. Altered Gut Microbiota in Irritable Bowel Syndrome and Its Association with Food Components. Journal of Personalized Medicine. 2021;11(1):35.

35.

Almeida

AAP

, Farah

, Silva

, et al. Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria. Journal of Agricultural and Food Chemistry. 2006;54(23):8738–43.

36.

Duffy

. Interactions mediating bacterial translocation in the immature intestine. The Journal of Nutrition. 2000;130(2):432S–436S.

37.

Lichtman

. Bacterial translocation in humans. Journal of Pediatric Gastroenterology and Nutrition. 2001;33(1):1–10.

38.

Berg

. Bacterial translocation from the gastrointestinal tract. Trends in Microbiology. 1995;3(4):149–54.

39.

Wiest

, Lawson

, Geuking

. Pathological bacterial translocation in liver cirrhosis. Journal of Hepatology. 2014;60(1):197–209.

40.

Murray

, Barbose

, Cobb

. Serum D (-)-lactate levels as a predictor of acute intestinal ischemia in a rat model. Journal of Surgical Research. 1993;54(5):507–9.

41.

Sobhian

, Kröpfl

, Hölzenbein

, et al. Increased circulating D-lactate levels predict risk of mortality after hemorrhage and surgical trauma in baboons. Shock. 2012;37(5):473–7.

42.

. Biomarkers of necrotising enterocolitis. In Seminars in Fetal and Neonatal Medicine. 2014:33–8.

43.

Sun

, Fu

, Rong-Zhang

, et al. Relationship between plasma D (-)-lactate and intestinal damage after severe injuries in rats. World Journal of Gastroenterology. 2001;7(4):555.

44.

Birkner

, Grucka-Mamczar

, Żwirska-Korczala

, et al. Influence of sodium fluoride and caffeine on the kidney function and free radical processes in that organ in adult rats. Biological Trace Element Research. 2006;109(1):35–47.

45.

, Han

, Kim

, Park

, Kim

, Lee

. Coffee consumption is associated with lower serum aminotransferases in the general Korean population and in those at high risk for hepatic disease. Asia Pac J Clin Nutr. 2016;25(4):767–75.

46.

Ding

, Zhang

. Associations of coffee consumption with the circulating level of alanine aminotransferase and aspartate aminotransferase. A meta-analysis of observational studies. Journal of the American College of Nutrition. 2021;40(3):261–72.

47.

Boekschoten

, Schouten

, Katan

. Coffee bean extracts rich and poor in Kahweol both give rise to elevation of liver enzymes in healthy volunteers. Nutr J. 2004;3(1):7.

48.

Urgert

, Meyboom

, Kuilman

, Rexwinkel

, Vissers

, Klerk

, Katan

. Comparison of effect of cafetiere and filtered coffee on serum concentrations of liver aminotransferases and lipids: six month randomised controlled trial. BMJ. 1996;313(7069):1362–6.

49.

Onuegbu

, Olisekodiaka

, Adebolu

, Adesiyan

, Ayodele

. Coffee consumption could affect the activity of some liver enzymes and other biochemical parameters in healthy drinkers. Med Princ Pract. 2011;20(6):514–18.

50.

Handayani

, Pratama

AGN

, Putra

IWA

. Effect of Ethanol Extract of Robusta Coffee (Coffea canephora) on the Function of the Liver and Kidney of Wistar Rat. In 12th Annual Scientific Meeting, Medical Faculty, Universitas Jenderal Achmad Yani, International Symposium on” Emergency Preparedness and Disaster Response during COVID 19 Pandemic”(ASMC 2021)) (pp. 67–71). Atlantis Press.

51.

Dianzani

, Muzio

, Biocca

, et al. Lipid peroxidation in fatty liver induced by caffeine in rats. International Journal of Tissue Reactions. 1991;13(2):79.

52.

Manne

, Saab

. Coffee as modulator of liver injury: Fact and fiction. Clinical Liver Disease. 2015;6(6):139.

53.

Fried

, O’Connell

. A comparison of the effects of prenatal exposure to tobacco, alcohol, cannabis and caffeine on birth size and subsequent growth. Neurotoxicology and Teratology. 1987;9(2):79–85.

54.

Nyblom

, Björnsson

, Simrén

, et al. The AST/ALT ratio as an indicator of cirrhosis in patients with PBC. Liver International. 2006;26(7):840–5.

55.

Amadi

, Agomuo

, Duru

MKC

. Toxicological studies of Asmina triloba leaves on haematology, liver, kidney using rat model. International Science Research Journal. 2013;4(2):11–7.

56.

Emmanuel

, Majesty

, Benjamin

, et al. Effect of caffeine on some selected biochemical parameters using rat model. Advances in Biology. 2017.

57.

Lushchak

. Contaminant-induced oxidative stress in fish: a mechanistic approach. Fish Physiology and Biochemistry. 2016;42(2):711–47.

58.

Djidel

, Bouaziz

, Bentehar

, et al. Effect of methanol extract prepared from leaf of Pistacia lentiscus on plasma antioxidant activity and biomarkers of oxidative stress in liver tissue of healthy rats. Annual Research & Review in Biology. 2018:1–10.

59.

Kamat

, Boloor

, Devasagayam

TPA

, et al. Differential modification by caffeine of oxygen-dependent and independent effects of γ-irradiation on rat liver mitochondria. International Journal of Radiation Biology. 2000;76(9):1281–8.

60.

Kriško

, Kveder

, Pifat

. Effect of caffeine on oxidation susceptibility of human plasma low density lipoproteins. Clinica Chimica Acta. 2005;355(1-2):47–53.

61.

Nikolic

, Bjelakovic

, Stojanovic

. Effect of caffeine on metabolism of L-arginine in the brain. In Guanidino Compounds in Biology and Medicine. 2003:125–8.

62.

Arslan

, Demir

, Ozbay

and Arslan

. Evaluation of Lipid Peroxidation and Some Antioxidant Activities in Patients with Primary and Metastatic Liver Cancer. Journal of Cancer Therapy. 2014;5(2):192–7.

63.

Abreu

, Silva-Oliveira

, Moraes

MFD

, Pereira

, Moraes-Santos

. Chronic coffee and caffeine ingestion effects on the cognitive function and antioxidant system of rat brains. Pharmacology Biochemistry and Behavior. 2011;99(4):659–64.

Caffeine citrate effects on gastrointestinal permeability,bacterial translocation and biochemical parameters in newborn rats after long-term oral administration

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Material and methods

2.1 Animals

2.2 Ethical considerations

2.3 Study design

2.4 Microbiota assessment

2.5 Assessment of bacterial translocation

2.6 Analysis of intestinal permeability

2.7 Serum biochemical parameters assays

2.8 Measurement of malondialdehyde (MDA) content, catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) activities in the ileum the and colon

2.9 Statistical analysis

3 Results

3.1 Effect of caffeine on body weight

Table 1 Bacterial translocation to MLNs, liver, spleen and thymus in treated and control groups Groups MLNs Liver Spleen Thymus 15 D CT 4/6 2/6 2/6 0/6 CF 2/6 1/6 0/6 0/6 30 D CT 2/6 2/6 1/6 0/6 CF 1/6 0/6 0/6 0/6 60 D CT 0/6 0/6 0/6 0/6 CF 0/6 0/6 0/6 0/6

Footnotes

Acknowledgments

Conflicts of interest

Funding

References

Table 1
Bacterial translocation to MLNs, liver, spleen and thymus in treated and control groups

Groups MLNs Liver Spleen Thymus

15 D CT 4/6 2/6 2/6 0/6

CF 2/6 1/6 0/6 0/6

30 D CT 2/6 2/6 1/6 0/6

CF 1/6 0/6 0/6 0/6

60 D CT 0/6 0/6 0/6 0/6

CF 0/6 0/6 0/6 0/6