The cocoa bean tree-Theobroma cacao (Linnaeus); family Sterculiaaceae, originated from Latin America about 500 years ago, from where it was domesticated in other parts of the world [1]. Harvested cocoa beans are usually fermented and dried prior to their processing into finished products [2,3]. Cocoa bean-beverages are processed products of the cocoa bean, sold under several brand names in Nigeria and worldwide [4-6]. The nutraceutical values of raw cocoa bean products (RCBP) [7-11] as well as the high acceptability of processed cocoa bean-based beverages (PCB-BB) [2], because of their attractive flavour and appearance, designate the cocoa tree as a highly prized international cash crop.

The quality parameters of PCB-BB in Nigeria markets have previously been reported elsewhere [5,6,12-14]. Previous studies have raised safety concerns about the consumption of RCBP- and industrial PCB-BB- containing diets. For the most part, the presence of anti-nutritional factors in RCBP is associated with the toxicological outcomes and poor nutritional score when used as feed substitutes for farm animals [15,16]. Likewise, the presence of Maillard reaction endproducts/ chemically modified by-products [2,17-21], heavy metal [22] and microbial contaminations [12,14,21-25] of PCB-BB- and RCBPcontaining diets may provoke tissue lesion and organ damage.

The liver and kidney are organs of homeostasis. The hepatic tissues play a central role in the biotransformation of xenobiotics and endogenous molecules prior to their elimination from the body [26-28]. The biotransformation of xenobiotics in the hepatocytes may elicit the formation of noxious and highly reactive compounds or potentially toxic metabolites, which in the process of their metabolism predisposes the hepatocytes to injuries and dysfunction [29]. The renal tissues are highly specialized in ensuring delicate balance in selective excreting or retention of body biomolecules according to their physiologic renal threshold indices [30]. The renal tissues are predisposed to chemical induced injuries because of their action to concentrate tubular fluid by removal of H₂O, organic compounds and inorganic salts from the vascular system. Liver (hepatic) function test (LFT) and renal function test (RFT) are diagnostic parameters for ascertaining organ integrity and functionality and level of recovery from pathologic injuries. Histopathological studies are precise methods for the identification and characterization of pathological changes associated with tissue lesion.

Chemical modifications of organic matters in cocoa bean occur through the processes of dextrinization, caramelization, pyrolysis, cyclization, oxidation and esterification reactions [19,20,31], which upon ingestion of the resultant organic derivatives may prompt tissue lesion in biologic systems. Additionally, studies have shown that farm and industrial PCB-BBs results in alterations of their physicochemical characteristics [2]. There is no available precise empirical information on the effect of PCB-BB - containing diets on internal organs integrity and functionality. Moreover, information on the effect of RCBP - containing diets on animal physiology is comparatively scanty and has been largely ignored and taken for granted. Accordingly, the present study ascertained functional integrity of renal and hepatic tissues of Wistar rats fed with PCB-BB - and RCBP - containing diets using biochemical and histological methods.

Collection and processing of raw cocoa-bean seeds

The cocoa-bean pods were randomly handpicked from smallholder cocoa farmers in Owerri, Imo State, Nigeria. The pods were harvested on the 24th September, 2014. The beans were evacuated from the pods and allowed to ferment for 5 days while shielded from sunlight. Fermentation of cocoa bean was done using the conventional heap fermentation method [32]. The wet beans were heaped on layers of plantain (Musa paradisiaca) leaves and covered with the same material to retain the heat generated during the fermentation process. On the third and fifth days, the beans were quickly and thoroughly re-mixed using a wooden spade and covered once again. Next, the fermented beans were sun-dried for ten days till constant weight was achieved. A 50 g sample of the beans were pulverized using Thomas-Willey milling machine (ASTM D-3182, INDIA), after which the ground samples were stored in air-tight plastic bottles with screw caps pending use to compound the rat diets.

Animal diets

The RCBP was mixed with sucrose (ratio 10:1 w/w) to sweeten it. The PCB-BBs were three (3) brands of cocoa beverages commonly consumed in Nigeria, which were purchased from a grocery shop. Also, PCB-BB and RCBP were compounded separately with PSGF (ratio 10:1 w/w) to obtain the test diets, whereas the control diet was composed of pelletized standard guinea feed (PSGF) only. The PSGF (product of a subsidiary of UAC Nigeria Plc., Jos, Nigeria) was purchased at the Relief Market, Owerri, Imo State, Nigeria.

Animal handling

The present study was approved by the Ethical Committee on the use of animals for the research, Department of Biochemistry, Federal University of Technology, Owerri, Nigeria. The rats were obtained from the Animal House of the Department of Biochemistry, Federal University of Technology, and Owerri, Nigeria. Female albino (Wistar) rats were maintained at room temperatures of 28 ± 2°C, 30–55% of relative humidity on a 12-h light/12-h dark cycle, with access to water and PSGF ad libitum for 2 weeks acclimatization period. Handling of the rats was in accordance with the standard principles of laboratory animal care of the United States National Institutes of Health.

Design of animal feed experiment

A total of 30 female Wistar rats (90 days old) of average weight of 106.0 ± 2.0 g were allotted into five (5) groups of six (6) rats each. The rats were deprived of food and water for additional 16 h before commencement of feeding as described elsewhere [33]. The rat groups were designated on the basis of experimental diets received for 28 days.

Group 1 (WR-PSGF): Wistar rats received PSGF + water ad libitum.

Group 2 (WR-RCBP): Wistar rats received RCBP + water ad libitum.

Group 3 (WR-OT): Wistar rats received OT + water ad libitum.

Group 4 (WR-BV): Wistar rats received BV + water ad libitum.

Group 5 (WR-MO): Wistar rats received MO + water ad libitum.

At the end of the feeding period, blood samples were drawn from the orbital sinus [34] of 12-hour post-fasted rat groups for renal and hepatic function tests. Also, renal and hepatic tissues were excised from the various rat groups for histological examinations.

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)

Measurement of serum AST and ALT activities were according to the methods of Reitman and Frankel, [35].

Bilirubin

Serum total bilirubin concentration (STBC) was measured using diazotized sulphanilic acid methods as previously described [36].

Total protein

Serum total protein concentration (STPC) was measured using the Biuret method as described by Gornall, [37].

Albumin

Measurement of serum albumin concentration (SAC) was by the method described by Doumas et al. [38].

Renal function test

Urea: Serum urea concentration (SUC) was measured using the rapid method as described by Fawcett and Scott, [39].

Creatinine: Measurement of serum creatinine concentration (SCC) was according to the methods as described by Bonsnes and Taussky [40].

Organ histology was according to the methods described by Banchroft [41]. Autopsy samples were taken from the renal and hepatic tissues of the different animal groups, fixed in 10% formolsaline (pH = 7.2) for 24 h and washed with continuous flow of distilled water. The specimens were cleared in xylene embedded in paraffin in hot air oven at 56 °C for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 4-mm thickness using a semi-automated rotatory microtome. The obtained tissue sections were collected on glass slides, dehydrated by immersing in serial dilutions of ethyl alcohol-water mixture, cleaned in xylene and embedded in paraffin wax. Next, the specimens were deparaffinized and stained with hematoxylin and eosin (H&E) dye for histopathological examinations. Photomicrographs of the tissue sections were captured using chare-couple device (CCD) camera under light microscope (Olympus BX51TF; Olympus Corporation, Tokyo, Japan) at × 400 magnification power.

Statistical analysis

The results were expressed as mean ± SEM, and statistically analyzed by one way ANOVA followed by Dunnett test, with level of significance set at p<0.05.

An overview of Figure 1 showed that levels of serum activities of the two LFT enzymes were in the order: ALT>AST. However, serum ALT activities of WR-PSGF, WR-RCBP, WR-OT, WR-BV and WR-MO showed no significant difference (p>0.05) and were within relatively narrow range of 32.17 ± 4.98 IU/L – 41.00 ± 10.85 IU/L.

Figure 1: Serum aspartate aminotransferase and alanine aminotransferase concentrations of experimental rat groups.

Conversely, serum AST activities of WR-PSGF, WR-RCBP, WROT, WR-BV and WR-MO gave wide variation, which was within the range of 15.67 ± 2.13 IU/L – 34.83 ± 8.31 IU/L; p<0.05. Specifically, WR-OT exhibited the lowest serum AST activity, whereas WR-RCBP gave the highest serum AST activity.

Generally, STBC of WR-PSGF, WR-RCBP, WR-OT, WR-BV and WR-MO were<1.0 mg/dL. WR-RCBP gave peak value of STBC = 0.85 ± 0.18 mg/dL, which was over 2 folds higher than that of WR-OT; STBC = 0.39 ± 0.04 mg/dL (Figure 2).

Figure 2: Serum bilirubin concentrations of experimental rat groups.

An overview of Figure 3 showed that STPC and SAC of WR-PSGF, WR-RCBP, WR-OT, WR-BV and WR-MO varied within relatively narrow range. Furthermore, STPC and SAC of WR-PSGF were not significantly different (p>0.05) from that of WR-RCBP. Finally, STPC and SAC of WR-OT, WR-BV and WR-MO were comparatively higher than those of WR-PSGF and WR-RCBP.

Figure 3: Serum total protein and albumin concentrations of experimental rat groups.

Figure 4 showed that WR-RCBP gave the highest SUC, which was significantly different (p<0.05) from that of other four experimental rat groups (WR-PSGF, WR-OT, WR-BV and WR-MO). Conversely, SUC of WR-PSGF, WR-OT, WR-BV and WR-MO showed no significant difference (p>0.05). Additionally, SCC was significantly lower (p<0.05) than SUC in WR-PSGF, WR-RCBP, WR-OT, WR-BV and WR-MO. Finally, SCCs in the five experimental rat groups were within relatively narrow range of 1.35 ± 0.23 mg/dL – 0.72 ± 0.03 mg/dL; p>0.05.

Figure 4: Serum concentrations of renal function indicators of experimental rat groups.

Hepatic parenchyma of WR-PSGF showed several hepatic lobules separated from each other by delicate connective tissue septa that served as repositories to the portal triad. Additionally, the hepatic lobules were consisted of thin walled central vein surrounded by hepatic cords with irregular blood spaces lined by endothelial cells and Von Kupffer cells. The nuclei appeared densely stained (Figure 5A). Also, renal tissues of WR-PSGF showed normal histology of renal corpuscles and tubules. The renal corpuscles were consisted of tuft of blood capillaries surrounded by the Bowman’s capsule (Figure 5B).

Figure 5: Photomicrograph of sections of organs from WR-PSGF. (A) Normal hepatic tissue with central vein (CV) and Kupffer cells along the sinusoids (blue arrow). (B) Renal tissue showing normal glomerulus (G) and renal tubules (blue arrows); H&E ×400.

Hepatic parenchyma of WR-MO showed normal architecture with thin walled central vein surrounded by hepatic cords. Additionally, the nuclei appeared densely stained. Renal tissue of WR-MO showed evidence of loss of cellular architecture.

Clinical surveys and animal model experiments have revealed that raised levels of ALT and AST activities are indicative of organ damage; specifically, in pathologic and toxicological events leading to cardiac and hepatic necrosis [29,42-44]. Precisely, earlier studies had associated raised serum ALT activity with non-diabetic non-alcoholic fatty liver disease and insulin resistance [45-50]. In the present study, the ratio of the two amino transferases activity in serum, whereby serum ALT>AST (Figure 1) was an indication that the two non-functional plasma enzymes were of hepatic origin rather than the cardiac tissues [48,51]. Accordingly, the pattern of activity of ALT>AST in serum appeared to correlate with the extent of disarrangement of hepatic tissue architecture following the consumption PCB-BB - and RCBP - containing diets by corresponding experimental rat groups. It is worthwhile to note that the remarkable histologic alterations in hepatic tissue architecture of WR-RCBP, WR-OT, WR-BV and WR-MO (Figures 6A-9A) were reflections of the significant (p<0.05) activity differentials between their ALT and AST values (Figure 1).

Figure 6: Photomicrograph of sections of organs from WR-RCBP. (A) Hepatic tissue with its central vein (CV) and Kupffer cells along the sinusoids (blue arrow). (B) Renal tissue showing venous congestion (VC); H&E ×400.

Figure 7: Photomicrograph of sections of organs from WR-OT. (A) Hepatic tissue showing the portal area (PA) with remarkable histologic change. (B) Renal tissue with moderate degenerated tubules and glomerular tufts (T); H&E ×400.

Figure 8: Photomicrograph of sections of organs from WR-BV. (A) Hepatic tissue showing central vein (CV) and moderate disarrangement of hepatocytes. (B) Renal tissue showing moderate hypercellularity of the glomerulus (G); H&E ×400.

Figure 9: Photomicrograph of sections of organs from WR-MO. (A) Hepatic tissue showing the central vein (CV) and normal plates of hepatocytes (blue arrows), (B) Renal tissue showing moderate congestion of the glomerulus (G) and interstitium (blue arrows); H&E ×400.

Previous reports have noted that human STBC>1.0 mg/dL – 1.2 mg/dL was diagnostic of hyperbilirubinemia [52,53]. The production of more bilirubin than the normal liver can excrete, failure of a damaged liver to excrete bilirubin produced in normal amounts and in the absence of hepatic damage, obstruction of the excretory ducts of the liver – by preventing the excretion of bilirubin are the underlying causative factors leading to the development of hyperbilirubinemia [52]. Studies have shown that ingestion of certain toxic food substances or compounds can provoke rapid erythrocyte haemolysis [54-57] and interfere with normal functions of the liver [57-60]. Accordingly, the peculiar proximate composition of RCBP as described elsewhere [2,7,8,61] may have caused the comparatively raised STBC in WR-RCBP following the consumption of RCBP - containing diet by the corresponding experimental rat group. Nevertheless, the results of the present study appeared to suggest the absence of hyperbilirubinemia in WR-RCBP, WR-OT, WR-BV and WR-MO in spite of the moderate histological changes in their hepatic tissues (Figures 6A-8A). It is worthwhile to note that bilirubin and its derivative –biliverdin, by virtue of their anti-oxidant activity, protects mammals against nephropathy, stroke, atherosclerosis and vasculitis [53,62-64] and bilirubin at micro-molar concentrations efficiently scavenged peroxyl radicals in vitro [53].

Although certain plasma proteins have their origin from the endothelial and plasma cells, most proteins biosynthesized in the hepatocytes eventually find their ways in plasma. Therefore, a compromised hepatic function engenders absence or low circulating levels of plasma proteins with attendant pathophysiologic conditions. The plasma proteins have been studied extensively in both humans and animals and the relationship between STPC/SAC and the nutritional status of humans are well established, as typified in cases such as marasmus and kwashiorkor [65]. Comparative assessments of STPC and SAC (Figure 3) did not suggest incident of poor nutritional status in the various experimental rat groups. Specifically, experimental rat groups fed with PCB-BB - containing diets exhibited relatively higher STPC and SAC than those fed with RCBP - containing diet and PSGF. By implication, feeding experimental rats with PCB-BB - and RCBP - containing diets satisfied the minimum physiologic nutritional standards required by the rats. Additionally, the PCBBB - and RCBP - containing diets - induced moderate changes in hepatic tissues histology of corresponding experimental rat groups did not substantially interfere with the capacity of the hepatocytes to biosynthesized plasma proteins.

Under normal physiologic conditions, urea is the primary vehicle for the excretion of metabolic nitrogen, whose sources are, for the most part, traceable to dietary constituents and body protein turnover [66,67]. Urea is a low threshold substance, which is why it is rapidly cleared from vascular system by the renal system. Therefore, raised level of blood urea nitrogen (BUN) concentration is diagnostic of renal dysfunction. The comparatively raised level of SUC in WR-RCBP (Figure 4) correlated with the structural alteration of the renal tissues as exemplified by the noticeable venous congestion of the tissue section (Figure 6B). However, the moderate degenerated tubules and glomerular tufts of WR-OT and WR-BV as well as moderate congestion of the glomerulus and interstitium of WR-MO did not profoundly affect the functionality of the renal tissues, since the SUC of the corresponding experimental rat groups were comparable with that of the WR-SGFP, whose renal tissue histology revealed normal glomerulus and renal tubules. Likewise, moderate disarrangement renal tissues of the various experimental rat groups did not adversely affect the capacity of their renal tissues to clear the blood of creatinine. Although the present study showed that SUC was greater than SCC in the experimental rat groups as described elsewhere [68-72], previous reports have shown that measurement of SCC offered a more reliable diagnostic parameter than SUC for confirmation of renal dysfunction [73,74]. Furthermore, Kang et al. [75] had earlier noted significant elevation of SUC as against marginal alterations of SCC in streptozotocin-induced diabetic rats that exhibited renal dysfunction, which conformed to the present findings (Figure 4). Creatinine is sourced from the muscle proteins turnover and urinary creatinine concentration is proportionate to muscle mass and remains relatively constant. Accordingly, the approximate equal body weights of the various experimental rat groups dictated the corresponding comparable SCCs of WR-PSGF, WR-RCBP, WROT, WR-BV and WR-MO. However, increase in SCC can result from increased ingestion of cooked meat [67].

The pattern of activity of ALT>AST in serum appeared to correlate with the extent of disarrangement of hepatic tissue architecture. The experimental rat groups did not exhibit hyperbilirubinemia. Also, PCB-BB - and RCBP - containing diets did not substantially interfere with the capacity of the hepatocytes to biosynthesized plasma proteins and the functionality of renal tissues.

The author is grateful for the technical assistance offered by Mr. O.A.K. Emenyonu, Chief Laboratory Technologist, Department of Biochemistry, Imo State University Owerri, and Nigeria.