A DEFICIENT RATS. A Introduction
The method developed for vitamin A administration in Trial IV was to prepare VAP Type 100 in dilute solution as a low-level vitamin A supplement. This would allow the maintenance of vitamin A-deficient rats in an otherwise normal condition. This supplement is administered in the drinking water (at a concentration of 0.1875 pgRE/ml when freshly made up) and replenished with each change of fresh water every 3 or 4 days.
The in vitro experiments (Trial V(a)) carried out by Roche Products Ltd. demonstrated that dilute solutions of VAP Type 100 in deionised water (0.3 pgRE and 3.00 pgRE/ml vitamin A
respectively) were substantially broken down over a 3-4 day period. The degree of VAP breakdown in the solution initially containing 0.3 pgRE/ml at time 0 was 31% after 24 hours, 49% after 48 hours, 59% after 72 hours and, by extrapolation, 60- 70% after 96 hours. A degree of VAP breakdown at least equivalent to those levels would therefore be expected over the 3 or 4 day periods of VAP administration to vitamin A-deficient rats in the animal holding room. The instability of VAP in the drinking water, coupled with the regular replenishment of water bottles with freshly prepared VAP solution, would thus be expected to cause the concentration of vitamin
A available for consumption by vitamin A-deficient rats to fluctuate in a cyclical manner.
The ejqDeriments described in this thesis rely upon the analysis of retinol levels in plasma samples taken at post-mortem to confirm the vitamin A status of rats fed a vitamin A-deficient diet. In the light of the in vitro studies demonstrating VAP instability in drinking water. Trial V(b) was carried out to investigate whether fluctuating VAP consumption would result in variations in the plasma retinol levels of vitamin A-deficient rats. This involved the daily determination of plasma
retinol levels in vitamin A-deficient rats over a period of 7 days (day 57 to day 64 of the
experiment), during which fresh VAP was administered in the drinking water on day 57 and day 6 r, respectively.
B Experimental Design
30 female F344 rats aged 18-21 days old were acclimatised for a period of 2 days, during which they were fed a standard laboratory maintenance diet in pelleted form. After acclimatisation, the rats were starved for 18 hours before being randomised by weight into 2 groups (day 0). The design of the trial is shown in Table 6.7. Group 1(12 rats) was fed the SSD(ii) diet plus a control
level of VAA (2064 ^igRE/kg). Group 2 (18 rats) was fed the SSD(ii) diet alone. From day 47, when demonstrating clinical signs of vitamin A deficiency, the rats in Group 2 were given VAP Type 100 in the drinking water. When freshly prepared, the concentration of vitamin A in this drinking water was 0.1875 pgRE/ml. The water was obtained from the mains supply. Fresh VAP was administered every 3 or 4 days, on days 47, 50, 54, 57 and 61 respectively.
The times of post-mortems are also shown in Table 6 .?. At post-mortem, plasma was prepared for retrospective analysis for vitamins A and E and for BC, and macroscopic findings related to vitamin A deficiency were noted. To examine the effect of fluctuating vitamin A intake (due to VAP breakdown in the drinking water) on plasma retinol levels, 2 or 3 rats from Group 2 (SSD(ii) diet alone) were killed on successive days after the administration of fresh VAP on day 57. At 24 hour intervals, plasma samples were taken from rats killed on days 57, 58, 59, 60 and 61. After the animals were killed on day 61, new drinking water, containing fresh VAP, was administered to the remaining rats, these being killed 2 days later on day 63. Animals fed the control diet (Group
1) were killed concurrently with vitamin A-deficient rats on days 57, 60, 63 and 64, respectively.
All rats were weighed regularly. From week 5, the animals were weighed each week and were examined in detail for clinical signs of vitamin A deficiency every working day. From week 4 until the end of the experiment, water consumption was monitored continuously over periods of 3 or 4 days.
C Results
(i) Body weights
The body weights of rats in Trial V(b) are presented in Figure 6.17. (The full tabulated data are shown in Table 6.8). At the start of the trial (week 0), the group mean body weights (g ± sd) were: Group 1 39.8 ±3.1 and Group 2 28.4 ± 2.9, respectively. The mean body weight of rats in Group 1 increased steadily over the first 5 weeks of the trial and continued to rise up to week 8, with the rate of gain slowing slightly over the later weeks. Rats in Group 2 gained weight at a similar rate to Group 1 over the first 3 weeks. However, between weeks 3-5 these animals demonstrated an appreciably slower rate of gain compared with those in Group 1, while from weeks 5-8 no further increase in weight was observed in Group 2 rats.
The large difference in mean body weight (11.4 g) between the two groups at the start of the trial was the result of the method used to allocate the individual rats to each group. Initially, a system of random numbers had been used; however, this method produced 2 groups which both
demonstrated very large ranges in body weight. If these groups had been used, individual rats would have become vitamin A-deficient at widely divergent times. Therefore, to induce the animals in Group 2 to become deficient more or less simultaneously, the rats in both groups were redistributed (before going onto their diets on day 0) according to weight. The heaviest rats were placed in Group 1 and the lighter c’^es (th . eater part of the batch) into Group 2. In this way, 2 groups were formed which differed markedly in mean body weight but in which the ranges of individual weights were very narrow, as indicated by the standard deviations of each group mean.
(ii) Clinical signs o f Vitamin A deficiency
The macroscopic findings at post-mortem in rats of Trial V(b) are shown in Table 6.9. All the rats (12/12) in Group 1 demonstrated a normal appearance. After laparotomy, all these animals were observed to possess very large abdominal fat depots. The intestines of these rats were normal, containing plenty of intestinal ingesta and showing no evidence of intestinal haemorrhage or swelling due to the build up of intestinal gas.
In contrast, more than half (10/18) of the rats in Group 2 demonstrated a range of clinical signs of vitamin A deficiency similar to that described previously in Trials II, III and IV. These clinical signs (see grading system described in Table 6.4) ranged from grade 1 (3 rats), through grades 2 (3 rats) and 3 (3 rats) to grade 4(1 rat). After laparotomy, the majority (16/18) of these animals were observed to possess no (3 rats) or negligible (13 rats) abdominal fat depots; the remaining 2 animals showed only moderate fat reserves. The 3 animals with no fet depots (classified as grades 2, 3 and 4 based on external appearance) also demonstrated intestinal gas; 2 of these animals also showed intestinal haemorrhage, the only 2 to do so.
To investigate whether vitamin A deficiency affected relative liver weight, the livers of animals from Groups 1 and 2 were weighed at post-mortem. The absolute and relative liver weights (g liver/kg rat body weight) are shown in Table 6.10. There was some evidence that the absolute liver weights of animals in Group 2 were lighter than those in Group 1. In contrast, livers from vitamin A-deficient rats (Group 2) did not show a reduction in relative liver weight, in comparison with control (Group 1) animals. This was because the body weights of animals in Group 1 were heavier than those of animals in group 2.
(Hi) Plasma retinol levels
The group mean plasma retinol levels of rats killed on day 57 to day 64 are shown in Figure 6.18 (For average plasma retinol values in each group on day 57 to day 64, refer to Table 6.11). On
day 57, the mean (± sd) plasma retinol levels (pg/dl) were; Group 1 (fed SSD(ii) plus VAA diet), 25.60 (± 0.62) and Group 2 (fed SSD(ii) diet plus VAP in the drinking water), 1.75 (± 0.21). The administration, on day 57, of fresh VAP to the rats remaining in Group 2 did not result in any marked differences in the mean plasma retinol values obtained on days 58, 59, 60 and 61.
Similarly, vdien fresh VAP was given the remaining animals on day 61, no significant effects were observed on the plasma retinol values obtained on day 63.
(iv) Water consumption
Group mean water consumptions, calculated as absolute values (g of water consumed/rat/day) and as values relative to body weight (g of water consumed/1 OOg rat body weight/day), from day 22 to the end of the experiment on day 64 are presented in Table 6.12. For the 4 day period of days 22-26, the mean (± sd) absolute water consumptions were: Group 1 (fed the SSD(ii) plus VAA diet), 22.6 (± 3 .5) g rat/day and Group 2 (fed the SSD(ii) diet plus VAP in the drinking water 16.0 (± 0.7) g rat/day. In general, the absolute water consumptions of the two groups remained roughly at these levels until the end of the experiment, with Group 2 animals drinking about 25% less than those in Group I .
For the 4 day period days 22-26, the mean (± sd) relative water consumptions were: Group 1, 201.7 (±31.6) g/body weight/day and Group 2 163.8 (± 7.6) g/lOOg body weight/day. With increasing body weight, the relative water consumption of rats in Group 1 steadily declined from that observed at days 22-26 to 108 .1 (± 14.1) g/lOOg body weight/day by day 64. Until days 44-47, a similar decline in the relative water consumption was observed in rats fed the vitamin A-deficient diet (Group 2). However, from day 47 to the end of the experiment, the period corresponding to the plateau phase of the body weight curve (Figure 6.17) and to the
administration of VAP (from day 47), the relative water consumption of rats in Group 2 rose slightly. At day 57-61, these animals drank 135.2 (± 28.6) g of water/kg body weight/day, a value which was 135% of the relative consumption of animals in Group 1.
(v) Estimated VAP intake
The average daily intake of VAP over each 3 or 4 day consumption period for rats in Group 2 is also shown in Table 6.12. (For full details of the method used to estimate VAP intake from the VAP degradation curve (Figure 6.15, dilution 2) and the absolute water consumption, refer to Appendix 2). From day 22-26 to the end of the experiment the estimated average daily VAP intake of rats in Group 2 ranged from 3 .69 - 5 .81 pgRE/rat/day.
D Conclusions
Trial V(b) was carried out to investigate whether fluctuating vitamin A intake (caused by VAP breakdown in the water bottles) would effect the plasma retinol levels of VAP-supplemented vitamin A-deficient rats. The experiment involved the daily determination of plasma retinol in deficient rats over a period of 7 days (days 57-64), during which fresh VAP was administered in the drinking water on days 57 and 61 respectively.
Despite a large difference in mean body weight at the start of the trial, a clear difference in the rate of body weight gain was discernible between control animals (Group 1) and Group 2 rats fed the vitamin A-deficient diet (Figure 6.17). Between weeks 5-8 the mean body weight of rats in Group 2 remained constant, showing the plateau characteristic of vitamin A deficiency. At week 8 (day 57), the presence of overt clinical signs and depressed plasma retinol values (Table 6.11)
confirmed that the rats in Group 2 were vitamin A-deficient.
After the administration of fresh VAP in the drinking water on days 57 and 61, no significant changes were seen in the mean plasma retinol value of rats in Group 2. Throughout the experimental period of day 57 - day 63, the mean plasma retinol value in these animals ranged between about 6-15% of the control values in Group 1. Thus, the administration of fresh VAP in the drinking water every 3 or 4 days did not significantly affect the plasma retinol levels of vitamin
A-deficient rats supplemented in this way, particularly when the standard deviations were taken into account (Figure 6.18).
Throughout the trial, control rats consumed about 23 ml/rat/day of drinking water. In contrast, vitamin A- deficient animals in Group 2 drank only about 16 ml/rat/day, or 25% less than the controls (Table 6.12). Estimated values for the daily VAP consumption of vitamin A-deficient rats ranged from 3 .69 - 5 .81 pgRE/rat/day. These values were calculated from the absolute water consumptions (Table 6.12) and the VAP breakdown curve (Figure 6.15, dilution 2). These estimated daily VAP intakes would provide more vitamin A than the 0.6 pgRE/rat/day which Cohn (1984) recommended as being the minimum required for the maintenance of vitamin
A-deficient rats. It is likely, therefore, that compared with the curve of VAP breakdown measured in Trial V(a), a higher degree of degradation took place under the experimental conditions of Trial V(b). A possible cause for this could be the continual displacement of drinking water by volumes of fresh air every time animals took a drink from the water bottles. This would provide additional oxygen and thereby potentiate further breakdown of the remaining VAP present in the drinking water.