Intestinal morphology assessments of rats fed phytic acid extract from sweet potato (Ipomoea batatas) and IP6

Authors


Correspondence

Dr Lowell Dilworth, PhD, Department of Basic Medical Sciences, Biochemistry Section, University of the West Indies, Mona Campus, Kingston 7, Jamaica.

Email: lowell.dilworth@gmail.com

ABSTRACT

Background and aim

Zinc deficiency due to low intake or unavailability by bioactive compounds may lead to morphological changes in the intestines resulting in disruptions in gut function. This study aims to assess effects of phytic acid on gut morphology of rats.

Methods

Diets supplemented with sweet potato phytate extract were fed to Wistar rats in zinc-deficient and zinc-sufficient states for 4 weeks. Similar test animals also had diets supplemented with the commercially available sodium phytate salt (IP6) for comparison. At the end of the feeding period, body weights, feed intake and markers of intestinal function were assessed.

Results

Acute zinc deficiency adversely affected the glycocalyx, goblet and Paneth cells within the small intestine. This may eventually lead to compromisation of the gut's immune system and further reductions in its metabolic and absorptive capacity. This was further aggravated by sweet potato phytate extract consumption. IP6 supplementation on the other hand, increased surface amplification in the jejunum resulting in increased gut transit time and more efficient absorption of nutrients.

Conclusions

To minimize compromisation of the gut's immune, absorptive and metabolic functions, adequate zinc supplementation is necessary especially if foods rich in phytates are included in the diet. Supplementation of the diet with IP6 seems to offset some of these effects with maximum benefits observed if the diet is properly supplemented with essential minerals.

INTRODUCTION

Inositol hexaphosphate (IP6) also called phytic acid, is a naturally occurring polyphosphorylated carbohydrate, abundantly present in many plant sources and in certain high-fiber sources, such as cereals and legumes.[1] Consumption of phytic acid-rich foods have been reported to reduce breast, colon and prostate cancers[2, 3] and it may have significant functions in signal transduction and cellular regulation.[4] Phytic acid has high affinity for divalent minerals particularly zinc, which is an essential mineral having critical roles in protein synthesis, maintenance of cells and neuronal cell development.[5] Phytic acid and its analogues are collectively referred to as phytate but in literature the term is mostly applied to the most abundant form, IP6.

It is suggested that at a phytate to zinc molar ratio of greater than 15:1, zinc may become unavailable for use by mammals.[6] Commonly consumed tuber crops including yams (Dioscorea sp.), dasheen (Colocasia esculenta) and sweet potato (Ipomoea batatas) were screened for zinc, phytic acid and phytate to zinc molar ratios. Of all the crops assessed, sweet potato had the highest phytate to zinc molar ratio (17:1) hence samples of this crop was chosen for this research. Sweet potato is a dicotyledonous plant belonging to the family Convulaceae. It is ranked as the seventh most important food crop globally and is grown in 111 countries.[7] It is a highly nutritious crop with high levels of ascorbic acid, starch, vitamins of the B complex and anthocyanins.[7, 8] Positive association between zinc status and goblet cells are well established, with zinc-deficient mice having poorly developed microvilli and microplicae on the ocular surface tissues and reduced numbers of goblet cells.[9] A zinc-finger transcription factor Klf4 (formerly GKLF) expressed in the epithelia of many organs is also shown to play an important role in proliferation and/or differentiation of many cells with specificity for goblet cells.[10] Paneth cells located at the base of the crypts of Lieberkühn within the gut, are the main sources of antimicrobial peptides in the small intestine, including human α-defensins HD5 and HD6.[11] These cells are known to concentrate zinc within their secretory granules and the numbers of these cells can be affected by manipulating zinc intake.[12] While phytic acid is known to chelate minerals, we are unsure if similar effects will be observed with consumption of phytate extract vs. commercially available sodium phytate salt (IP6) in the zinc-sufficient and deficient states. In this study, goblet cells and Paneth cells, which are influenced by zinc levels, were assessed, along with intestinal morphology.

METHODS

Fresh matured sweet potato (Ipomoea batatas) tubers were harvested from a local farm in central Jamaica. Samples were washed with distilled water, oven dried to constant weight and ground into fine powder.

Extraction and determination of phytic acid

Phytic acid was extracted by a modification of the method of Samotus and Schwimmer.[13] Sweet potato was blended with 10% tri-chloro acetic acid (TCA) in a Waring Blender. The slurry was filtered in a sintered glass funnel and the residue washed successively with known volumes of 5% TCA. Filtrates were combined, neutralized with NaOH and freeze-dried for use as phytic acid extract. Phytic acid in the extract was determined by the method of Holt[14] as described by Davies and Olpin.[15] Commercial sodium phytate was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Feeding experiments

The experimental animals were 36 adult Wistar rats, which were assigned by weight into six groups (Table 1) of six rats each; average body weights 236.4 g. Adult rats (3 months old) were chosen for this study. Diets were prepared according to standard methods of diet preparations, AIN-93G purified rodent diet. AIN-93G vitamin mix and AIN-93G mineral mix (Dyets, Bethlehem, PA, USA).[16] Control diets fed to groups D and D + Zn were formulated without or with zinc supplementation, respectively. Diets D + Zn + PE and D + Zn + IP6 were formulated to simulate the level of phytate to zinc ratio (18:1) observed in sweet potato commonly consumed in the Caribbean. Diets prepared for Groups D + PE and D + IP6 had phytate extract from sweet potato and IP6 supplementation, respectively, without zinc, in order to assess the effects of phytate on low dietary zinc intake. Zinc was added as part of the mineral mix to the diets of groups D + Zn, D + Zn + PE and D + Zn + IP6 animals, in the form of zinc carbonate at a concentration of 1.65 mg/kg.[17] Zinc in diets D, D + PE and D + IP6 were negligible as determined by atomic absorption spectrophotometry. The respective diets are listed in Table 1.

Table 1. Test groups and their respective diets
GroupsDiet CompositionGroup names
1Formulated dietD
2Formulated diet + ZnD + Zn
3Formulated diet + Zn + phytic acid extractD + Zn + PE
4Formulated diet + Zn + IP6D + Zn + IP6
5Formulated diet + phytic acid extractD + PE
6Formulated diet + IP6D + IP6

Rats were housed in stainless steel cages in a room kept on a 12 h light-dark cycle, and allowed access to their respective diets and water freely. All animals acclimatized to the new diet and then fed their respective test diets for 3 weeks. Body weight changes and total food intake were recorded. Rats were killed by decapitation following an overnight fast. Portions of the upper jejunum were used for this study. Approval for the study was obtained after presentation of the protocol to the Board of the Department of Basic Medical Sciences, University of the West Indies, Mona Campus, Jamaica. Feed efficiency ratios were calculated as cumulative results of average feed intake per rat per week divided by weekly changes in body weight.

Histology and cell counting

Following death, jejuna portions were removed, fixed in 4% paraformaldehyde then embedded in paraffin. Sections were deparaffinized in xylene, then rehydrated in ethanol and stained with either hematoxylin and eosin or alcian blue periodic acid schiff (PAS) staining as previously described.[17, 18] Alcian blue was used to determine acid mucins while neutral mucins were determined following PAS-alcian blue staining. Paneth cells were also assessed following this method. After staining, sections were dehydrated in ethanol, cleared with xylene and mounted with cytoseal 60 (Stephens Scientific, Riverdale, NJ, USA). Images were captured on a Leica DMRME research microscope equipped with a DC 500 digital camera (Wetzlar, Germany). Villus height, villus width and crypt depth were measured. Width of the glycocalyx and epithelial cells were measured at ×1,000 using oil emersion lens. The ratio of goblet cells to total epithelial cells was obtained by counting, in a blinded fashion, total epithelial cells and alcian blue PAS positive cells with goblet morphology from 10 high-powered fields of well-oriented cross-sections.[19] Paneth cells were also identified from these samples.

RESULTS

Fig. 1 shows the effects of phytic acid and zinc supplementation on villus height, villus width and crypt depth. The data shows that the effects of phytic acid on villus height depends on the source of the supplement. Both test groups with diets supplemented with IP6 had significantly longer villi compared to all other groups. Conversely, test animals fed diets supplemented with sweet potato phytate extract (D + Zn + PE and D + PE) had significantly shorter villi compared to all other groups. These observations were also highlighted in Fig. 2 as villi heights of animals fed the control diet (Fig. 2b) were compared to those of test animals fed a prepared diet along with sweet potato phytate extract only (Fig. 2a). Reductions in villi heights were observed in test animals fed diets supplemented with phytae extract (D + Zn + PE and D + PE) compared to those fed the control diet (D + Zn). It was observed that animals in the test group D + Zn + IP6, had significantly greater villi width compared with all other groups while crypt depth values were highest in rats with zinc and sweet potato phytate extract added to the diets.

Figure 1.

Villus height, villus width, and crypt depth of the upper jejunum of rats fed diets containing phytate extract or IP6 with or without zinc supplementation. In assessing a specific parameter, different letter subscripts assigned to separate groups indicate significance difference (< 0.05). Values are expressed as mean ± standard error of the mean (SEM) where n = 10. All parameters were assessed at magnification ×1,000. Significantly reduced villus heights were recorded for both test groups fed diets supplemented with sweet potato phytate compared to all other groups. Crypt depth was only significantly decreased in group D + Zn. Villus width was significantly increased in rats fed diets supplemented with zinc and IP6.

Figure 2.

Cross sectional view of jejuna of rats fed sweet potato phytate extract compared to normal rats. Panel (a) shows hematoxylin and eosin (H&E) stained transverse section view of jejunum of rats fed diets supplemented with sweet potato phytate extract (gp D + PE) at ×10 magnification. Significantly reduced villi heights are noted in this test group compared to the control animals. Panel (b) shows H&E stained transverse section view of rat jejunum of the control group (D + Zn) at ×10 magnification. Villi heights were significantly increased in this control group (D + Zn) compared to rats fed phytate extract.

Table 2 shows that final body weights were significantly reduced in rats fed phytate extract without zinc (D + PE) compared to the groups fed diets with and without zinc supplementation (D and D + Zn), or commercial phytate plus zinc (D + Zn + IP6). Daily food intakes among the groups were not significantly different. Feed efficiency ratios were highest in animals that had commercial phytates and zinc (D + Zn + IP6) added to the diet. This was followed by the control group that had zinc supplemented diets (D + Zn). The lowest levels of feed efficiency were observed in groups D + PE and D + IP6, which had phytate extract and IP6 added to the diets, respectively.

Table 2. Food intake, body weights and feed efficiency of rats fed IP6, phytic acid extract as well as normal diets
Test groupsFinal body weight (g)Initial body weight (g)Daily feed intake/rat (g)Feed efficiency ratios
  1. Initial body weights were measured at the start of the experiment, while final body weights were taken on the last day. Feed efficiency ratios were calculated as cumulative results of average feed intake per rat per week divided by weekly changes in body weight.

  2. For each variable assessed, values in the same column with different subscripts are significantly different (P < 0.05). Values with different letter subscripts are also significantly different. Values are expressed as mean ± SEM where = 4.

Diet (D)256.3 ± 23.9a236.8 ± 16.512.1 ± 1.10.2 ± 0.3
D + Zn260.9 ± 19.3a236.5 ± 14.511.9 ± 0.80.8 ± 2.2
D + Zn + PE215.6 ± 14.2ab236.3 ± 14.98.1 ± 1.40.1 ± 0.4
D + Zn + IP6250.0 ± 21.1a236.5 ± 21.211.4 ± 0.84.9 ± 3.8
D + PE151.1 ± 16.4b236.0 ± 15.75.9 ± 0.4−0.2 ± 0.1
D + IP6241.4 ± 17.1ab236.7 ± 16.513.4 ± 0.8−0.1 ± 1.7

Fig. 3 shows transverse section view of jujena of rats fed a zinc-deficient diet compared to those fed the control diet. The alcian blue staining clearly highlights acid mucins within goblet cells. Reduced numbers of goblet cells were observed in jujena of rats that consumed zinc-deficient diets compared to rats fed zinc-sufficient diets.

Figure 3.

Histological sections of Wistar rat jejuna highlighting goblet cells. Alcian blue stained acid mucins within goblet cells are clearly visible at magnification ×20. Lower numbers of goblet cells were recorded in the jejuna of rats fed zinc-deficient diets (a) compared to the control group (b).

Fig. 4 shows the effects of phytate extract and IP6 supplementation on villus height to crypt depth ratio in the upper jejunum of Wistar rats. Villus height to crypt depth ratios were significantly higher in rats fed diets supplemented with zinc and IP6 compared to those fed diets supplemented with zinc and sweet potato phytate extract. Other groups including control and test groups did not show significant differences in villus height to crypt depth ratios.

Figure 4.

The effects of phytate extract and IP6 supplementation on villus height to crypt depth ratio in the upper jejunum of Wistar rats. Different letters or letter combinations assigned to different columns indicate significant difference among study groups at the P < 0.05 level. Measurements of villus height and crypt depth were taken from 10 fields of view. Values are therefore reported as mean ± standard error of the mean (SEM) where n = 10. Rats fed zinc-supplemented diets supplemented with sweet potato phytate extract displayed significant reductions in villus height to crypt depth ratio compared to those fed zinc supplements along with IP6.

Fig. 5 shows effects of phytic acid and zinc supplementation on Paneth cells as well as goblet cell numbers. All experimental groups fed zinc supplemented diets recorded higher goblet cell numbers compared to test animals that were fed zinc-deficient diets. These results were corroborated by a comparison outlined in Fig. 3 where there were observed reductions in goblet cell numbers in rats fed zinc-deficient diets compared to rats given zinc-sufficient diets. A similar observation was made in assessing Paneth cells, as all test animals fed zinc supplemented diets recorded higher Paneth cell numbers (Fig. 5) compared to those fed diets without zinc supplementation. This observation was made irrespective of whether or not the diets were additionally supplemented with IP6 or sweet potato phytate extract. A direct relationship between zinc supplementation and Paneth and goblet cell numbers was established as the highest numbers of these cells were observed in all animals fed zinc supplemented diets.

Figure 5.

The effects of phytate extract, IP6 and zinc supplementation on Paneth cells and goblet cell numbers in Wistar rats. Values are reported as mean ± standard error of the mean (SEM) where n = 10. All groups fed zinc-supplemented diets recorded higher goblet cell and Paneth cell numbers compared to zinc-deficient diets. Similar subscript symbols ascribed to different columns representing different test groups indicate no significant difference among the groups at the P < 0.05 level. Data in this figure confirm the direct relationship between zinc content of the diets and goblet as well as Paneth cell numbers.

Table 3 shows that zinc supplementation had a positive effect on the width of the glycocalyx as all test animals with zinc-supplemented diets had thicker glycocalyces compared to those without zinc supplementation. This was most noticeable in animals from the control group (D + Zn) as well as those with diets supplemented with zinc and sweet potato phytate extract (D + Zn + PE) with both groups displaying significantly thicker glycocalyces compared to all other test animals. Intestinal epithelial cell width of all test groups is also highlighted in Table 3. Rats fed diets supplemented with zinc and IP6 (D + Zn + IP6) recorded significantly higher epithelial cell width compared to those fed diets without zinc supplementation (D) or those fed diets supplemented with sweet potato phytate (D + PE).

Table 3. The effect of zinc and phytate supplemented diets on width of the intestinal epithelial cells and glycocalyx
GroupsEpithelial cell width (μm)Glycocalyx thickness (μm)
  1. Animals in group D + PE recorded significantly reduced glycocalyx thickness as well as lower epithelial cell width compared to other test animals.

  2. †,‡

    For either variable assessed, values in the same column with different subscripts are significantly different (P < 0.05). Values with different letter subscripts are also significantly different. Values are expressed as mean ± standard error of the mean (SEM) where n = 4.

D27.89 ± 0.87b24.11
D + Zn29.94 ± 0.52ab33.09
D + Zn + PE31.07 ± 1.07ab32.95
D + Zn + IP632.87 ± 0.99a25.83
D + PE27.37 ± 1.08b17.41c
D + IP630.66 ± 1.69ab23.02

DISCUSSION

As outlined in Table 1, the most significant weight gain was observed in the control group fed formulated diet and Zinc (D + Zn). Weight gain was also observed in the group fed a formulated diet only, as well as in the groups that had IP6 supplemented diets. Weight loss was recorded in the two groups fed diets supplemented with sweet potato phytic acid extract. This may be due to reduced food intake as a result of altered consistency and reduced palatability of the formulated diet following addition of the extract. Reduced palatability of the extract compared to the standard diet may also explain the reduced feed efficiency ratio in rats fed prepared diet and phytate extract only.

Intestinal villus and crypt parameters

The significant increases in jejuna villi heights observed in groups D + Zn + IP6 and D + IP6 resulted in rats belonging to these two groups displaying greater surface amplification compared to others (Fig. 1). This increased intestinal surface amplification resulted in increased surface area for the formation of the glycocalyx leading to more efficient nutrient absorption.[13] Villi height increase could also be a natural compensatory response to reduced nutrient availability. This compensation was effective as reflected in the percentage weight gain exhibited by rats given IP6 with and without zinc supplementation (5.71% and 1.98%, respectively). On the other hand, as outlined in Fig. 1, significant reductions in villi height as was observed in both groups fed phytate extract, ultimately resulted in reduced surface amplification. This in turn resulted in reduced absorptive capabilities of the intestines. This occurs as a result of significant fall-offs in mucus production due to a reduced goblet cell numbers.[10] This may also be due to a reduction in the number of absorptive enterocytes, a direct result of reduced villi height. This decreased absorptive capability may lead to reduced gut transit times and eventual weight loss. This may be one of the factors that contributed to the weight loss observed in rats fed phytate extract (Table 1). The significant reduction in villi length observed in these two groups may also be as a result of lowered permeability of the intestines to nutrients[20] or overall lack of nutrients arising from antinutrient factors within the extract. Data outlined in Fig. 1 also indicate that the highest crypt depth values were observed in groups D and D + Zn + PE, respectively. This is indicative of increased mitotic activity within the intestinal crypts resulting in increased numbers of precursor cells available to replenish the villi.[21] This again may also be geared at increasing the surface area of the villi for enhanced nutrient absorption capacity. Consequently, low crypt depths resulted in decreased mitotic activity within the intestinal gland where precursor cells exist. This ultimately results in a reduction in the ability to replenish cells in the villi. Reductions in crypt depth may therefore be reflected in reductions in villi height. This observation of villi height being directly related to the crypt depth, was made in all groups except group D + Zn + PE (Fig. 1).

Reduced villus height to crypt depth ratio is suggestive of reduced overall capacity for digestion and absorption of nutrients. As outlined in Fig. 4, test animals that were fed diets supplemented with sweet potato phytic acid extract along with zinc, had the lowest villus height to crypt depth ratio, reconfirming that these rats experienced a diminished capacity to absorb nutrients. Animals from the control group along with those that had IP6 supplemented diets displayed the greatest villus height to crypt depth ratios. This is a further indication that IP6 positively contributes to the increased overall capacity for nutrient digestion and absorption.

Glycocalyx

The glycocalyx is in essence, an outer layer on the cell membrane with bound glycoproteins and glycolipids.[22] It lines the small intestines where it incorporates a variety of digestive enzymes and has a pivotal role in the absorption of nutrients.[23, 24] Rats were fed zinc-supplemented diets had thicker glycocalyces compared to those fed zinc-deficient diets (Table 3). This also suggests that zinc deficiency has a greater impact than phytate extract on the glycocalyx as animals belonging to group D + Zn + PE had significantly thicker glycocalyces compared to those in group D + PE. This trend was not observed with IP6 treatment as values in those test groups did not vary significantly irrespective of zinc status. Interestingly, zinc and phytic acid extract supplementation together, had a positive effect on the glycocalyx. This was not previously recorded in literature; hence further investigations are required to unravel the underlying mechanisms. A diminution of the glycocalyx was observed in group D + PE. This is due mainly to the observed paucity of the villi as observed in Fig. 2a (D + PE) compared to control (D + Zn) (Fig. 2b). This may be the main contributory factor to the significant weight loss observed in the rats from test group D + PE. The reasoning is that diminution of the glycocalyx will have a direct negative effect on digestion since the absorption of nutrients in the affected rats will be compromised, a direct result of reduced surface amplification. Young and Heath,[24] suggested that enzymes which hydrolyze disaccharides and dipeptides are not only present in the apical cytoplasm of the intestinal cells and microvilli of the epithelial cells but are also released and absorbed unto the filaments of glycocalyx. This indicates that the glycocalyx is a “hot bed” of enzymatic activity, most of which are involved in the digestion and absorption of food. Changes in the microvillus-glycocalyx compartment can therefore affect the potential for incorporation of membrane-associated hydrolytic enzymes and transporter molecules, which are key aspects of nutrient metabolism.[25] Fawcett and Jensh,[21] also suggested that the presence of numerous microvilli, which are encapsulated by the glycocalyx, also contributes to the surface amplification of the intestines. Reduced microvilli and glycocalyx by extension results in diminished capacity to absorb nutrients. Supplementation of the diet with the phytate extract but without adequate zinc may therefore contribute to weight loss but the paucity of villi observed in group D + PE (Fig. 2a) may also be indicative of cytotoxic effects of the crude extract components in the absence of zinc. This is certainly not a conclusion however, as the enterocyte mitotic rate is probably the most sensitive index of intestinal pathology,[26] but this parameter was not assessed. This observation strongly indicates that adequate supplementation of the diet with zinc is necessary for the production and maintenance of the glycocalyx.

Goblet cells

The data outlined in Fig. 5 for total goblet cell count provides a clear indication of the effects of zinc supplementation on goblet cells as the numbers were higher in zinc supplemented groups compared to rats fed zinc-deficient diets. The lowest goblet cell numbers were observed in groups fed diets supplemented with phytic acid extract. This group displayed goblet cell numbers that were even lower than the values recorded for animals in test group D that were fed a zinc-deficient diet only. This reiterates the importance of zinc supplementation in high phytate diets. Goblet cells elaborate mucins, trefoil proteins and other factors that help protect the intestinal mucosa from injury and facilitate tissue repair.[27] This mucus gel layer is an integral structural component of the intestine acting as a medium for protection, lubrication and transport between the luminal contents and the epithelial cell lining.[28] Reduction in zinc levels therefore results in reduced mitotic activity within the intestinal gland where all goblet precursor cells are produced.[29] Further research aimed at assessing goblet cell numbers in the large intestine and the effects of plasma zinc concentration is therefore needed. This is because the large intestine is the limiting factor that determines transit time of food.

Paneth cells

Quantification of Paneth cells gave significantly higher values in the zinc supplemented controls compared to all other groups (Fig. 5). The lowest Paneth cell numbers were recorded in the zinc-deficient groups fed phytate extract and IP6. This is consistent with findings of Bevins,[30] who recorded increased Paneth cell numbers in zinc-sufficient rats. Other studies,[29] have also illustrated an increase in crypt cell production rate in the small intestine of the zinc-supplemented mouse. Lower dietary zinc will therefore lead to reductions in Paneth cell numbers owing to reduced mitotic activity within the intestinal glands where all precursor cells occur. If this situation persists, the immune defense capabilities of the gut will eventually be compromised since these specialized cells contain lysozyme, which digests bacterial cell walls and also secrete immunoglobulins and polypeptide antibiotics that destroy parasites and bacteria.[23, 31] Granule secretion is therefore regulated by the presence of bacterial flora.

Hypertrophy parameters

The significant increase in jejunal villus width observed in group D + Zn + IP6 may be due to cellular hypertrophy (Fig. 1). This is corroborated by the observation that the highest epithelial cell width was observed in rats from this test group (Table 3). This suggests that supplementation of the diet with phytic extract or IP6 may be responsible for mild intestinal hypertrophy resulting from increased workload.

The observations from this study show that intestinal morphology and function in rats is adversely affected by zinc-deficient diets. The effects may be made worse by a crude phytate extract. Zinc supplementation is necessary for the proper maintenance and functioning of the jejuna glycocalyx, goblet cells and precursor stem cells. Paneth cells, which have a defensive role in the intestines may be negatively affected by low zinc levels exacerbated by inadequate supplementation of diets containing phytate extract. Therefore, to minimize immune compromisation of the gut, adequate zinc supplementation is necessary, especially if high phytate diets are consumed. This research shows that further detailed studies assessing the mechanisms behind the combined positive effects of zinc and phytate supplementation on the glycocalyx are required. The recorded alterations of jejuna morphology by phytic acid resulted in increased surface amplification, which may increase gut transit time and decreased rate of nutrient absorption. This may be useful for people with diabetes who desire slow rates of nutrient absorption to prevent a spike in blood glucose. Other alterations in intestinal morphology following phytate supplementation may be geared towards compensating for reduced nutrient intake.

ACKNOWLEDGMENTS

This study was supported by a Postgraduate Research and Publications grant from the University of The West Indies, Mona Campus. We are grateful to Mr Haynes and Ms Johnson in the Anatomy Section of Basic Medical Sciences, UWI Mona for their technical help. The authors are also grateful to Ms Sanette Hall for her editorial input.

Ancillary