CULTURA, GLOBALIZACIÓN E INTERCULTURALIDAD

A Introduction

In Trial IV, it was shown that low-levei VAP given to rats in the drinking water from the mains supply was capable of maintaining vitamin A-deficient animals in an otherwise healthy condition for up to 16 weeks. However, of the 6 different dose levels examined, only the highest (3.0 pgRE/rat/day) reversed all clinical signs of deficiency and permitted the survival of animals with plasma retinol levels at about 20% of normal control values. In contrast, the lower dose levels of VAP, ranging from 0.03 to 1.5 pgRE/rat/day, all failed to maintain survival of vitamin A-deficient rats beyond about 8 weeks from the start of VAP administration. These results were une?q)ected, as the level of vitamin A required to maintain adult rats in a vitamin A-deficient state was reported to be 0.6 pgRE/rat/day (Cohn 1984). The findings, therefore, suggested that the VAP in the

drinking water was undergoing considerable degradation during each 3 or 4 day VAP

administration period (the time periods between the refilling of water bottles with fresh drinking water).

To investigate this possibility, a series of in vitro experiments were carried at Roche Products Ltd. (Putnam 1985) to examine the stability of VAP in dilute aqueous solution. Two experiments were conducted, both of which were performed over a 72 hour period, equivalent to a 3 day VAP administration period. In the first experiment, the stability of VAP Type 100 in fresh deionised water was determined at starting concentrations of 0.3 pgRE/ml and 3.0 pgRE/ml. The second experiment involved investigating any differences befveen fresh deionised water and fresh glass distilled water with respect to the stability of VAP in Rovisol Type 100, a commercial mixture of vitamins A, D and E (Roche Products Ltd.). In this experiment the starting concentration of the VAP in the Rovisol Type 100 solutions was 300 pgRE/ml (1000 iu/ml).

B Materials and Methods (i) Preparation o f samples a VAP Type 100

A 0.1 g sample of VAP Type 100 was added to 1000 ml of freshly prepared deionised water (dilution 1; vitamin A concentration: 3 .0 pgRE/ml). 25 ml of dilution 1 was added to 225 ml of fresh deionised water (dilution 2; vitamin A concentration: 0.3 pgRE/ml). A total of 6 ahquots of each dilution was prepared, to provide samples for the determination of VAP concentration at each of 6 time points.

b Rovisol Type 100

100 ml solutions containing Ig of Rovisol were prepared with fresh deionised water or fresh glass distilled water. Each solution contained vitamin A at a concentration of 300 pgRE/ml.

For UV spectrophotometry, 1 ml of each 1% Rovisol soluti^ i was diluted to 100 ml with

deionised or glass distilled water to give a final concentration of 3.0 pgRE/ml. This 3.0 pgRE/ml solution was freshly prepared before each determination.

fii) Experimental Design

All solutions were left standing at room temperature (22°C) in non-actinic glassware. The concentration of vitamin A in each solution was determined at time 0, 12, 24, 36 and 48 and 72 hours using UV spectrophotometry.

C Results

The results of the experiment to assess the stability of VAP in dilute solutions of VAP Type 100 in deionised water are presented in Figure 6.15 (for the raw data, please refer to Table 6.5). Both of the two dilutions of VAP Type 100 examined (i.e. VAP at initial concentrations of 3.0 pgRE/ml [dilution 1] and 0.3 pgRE/ml respectively [dilution 2]) demonstrated a fall in the concentration of vitamin A palmitate over the 72 hour test period. In both cases, the fall in concentration was (Figure 6.15) steepest over the first 24 hours and then became more shallow over the next 48 hours. The more dilute solution, dilution 2, demonstrated the greater reduction in concentration falling from 0.28 pgRE/ml at time 0 to 0.17 pgRE/ml after 12 hours, a decrease of 39%. After 72 hours, the concentration of VAP in this dilution had fallen to 0.11 pgRE/ml, a total decrease of 59%. In contrast, the concentration of VAP in the other solution (dilution 1) fell from 3.07 pgRE/ml to 2.26 pgRE/ml over the first 12 hours, a fall of 26.5%, Wiile the concentration after 72 hours was 1.46 pgRE/ml, a total decrease of 52%.

The results of the experiment to assess the effect of deionised water or glass distilled water upon the stability of VAP in solutions of Rovisol Type 100 are shown in Figure 6.16 (for the raw data, please refer to Table 6.6). Over the 72 hour test period, there was no significant difference in the concentration of VAP between the solution made up in deionised water and that in glass distilled water. After 72 hours, the concentration in the solution made with deionised water had fallen from

3.13 pgRE/ml at time 0 to 2.6 pgRE/ml, a total reduction of 17%. Likewise, the concentration in the solution made with glass distilled water had fallen by 16%, from 3.07 pgRE/ml at time 0 to 2.59 pgRE/ml after 72 hours.

D Conclusions

The stability over 72 hours of dilute aqueous solutions of VAP, as VAP Type 100, in deionised water was investigated (Figure 6 .15). Additionally, the effect of deionised water or glass distilled water was investigated upon the stability of VAP in solutions of Rovisol Type 100 (Figure 6.16).

VAP was unstable in very dilute solutions of VAP Type 100 in deionised water. After 12 hours, the concentration of the solution initially diluted to give 0.3 pgRE/ml, had fallen by 39%, while the more concentrated solution (diluted to give 3.0 pgRE/ml) had fallen by almost 27%.

Furthermore, deterioration in the vitamin A absorbance curve, suggesting a chemical change, was very noticeable (Table 6.5). This was especially noticeable in the 0.3 pgRE/ml dilution, with the UV maximum changing from 328 nm to 314 nm after 12 hours. The higher concentration did not show UV changes until the vitamin A determination at 36 hours.

The water, whether deionised or glass distilled, made little difference to the stability of VAP in solution of Rovisol Type 100 over 72 hours. In contrast to the first experiment involving VAP Type 100, it was noticed that when more concentrated solutions of Rovisol (300 pgRE/ml VAP) were kept and diluted immediately prior to each spectrophotometric determination, the reductions in vitamin A content were much lower. After 12 hours, the reduction was between 3-4% while after 72 hours it was only between 16-17%, with no deterioration in the maximum UV absorbance of vitamin A (UV max.) being detected. This greater stability of VAP in the solutions of Rovisol was probably due to a concentration effect and not to the vitamin E present in Rovisol Type 100. The antioxidant activity of vitamin E is associated with the alcohol form a-tocopherol, and not to the ester, a-tocopherol acetate, the form present in Rovisol Type 100 (Putnam 1985).

Furthermore, a concentration effect was observed in the experiment with VAP Type 100, with the lower concentration being less stable than the higher concentration. The cause of these

concentration effects is uncertain, but it may be related to the relative proportionality between palmitate molecules and active oxygen atoms. It is noteworthy that both VAP Type 100 and

Rovisol Type 100 contain the antioxidants BHA and BHT, in the absence of which the fall in VAP concentrations would have been, no doubt, even greater.

(i) Relevance o f these findings to the maintenance o f vitamin A-deficient rats

The findings of this in vitro investigation of the stability of VAP in dilute solution have clear implications for the studies described in this thesis in which low-level VAP supplementation was used for the long-term maintenance of vitamin A-deficient rats. Vitamin A-deficient animals are maintained by the administration of VAP in the drinking water such that, if no VAP degradation

occurred, each rat would consume 3 .0 pgRE/ml each day. To do this, VAP type 100 was diluted in deionised water to give a concentration in the water bottles of 0.1875 pgRE/ml. In the light of the present in vitro investigation, and given that water from the mains supply was used, it is clear that the concentration of VAP available in the water bottles for consumption would fall by at least

59% over 72 hours.

Extrapolation of the VAP degradation curve (Figure 6.15, dilution 2) from 72 hours to 96 hours would suggest that over a 4 day period the total fall in the VAP concentration in vitro could be between 59% to 70%.

In the absence of any degradation, rats administered a solution of VAP containing 0.1875 |igR£/ml would consume a theoretical total of 9.0 pgRE in 3 days or 12.0 pgRE in 4 days. In reality, however, the total VAP consumptions are estimated to be, at the most, 5.4 pgRE in 3 days and between 4.8-6.0 pgRE in 4 days. Based on these estimated figures, each rat would consume an average of 1.8 pgRE/day in every 3 day VAP administration period or 1.35 pgRE/day in every 4 day period. The method used to estimate the intake of vitamin A from the amount of water consumed and the VAP breakdown curve (Figure 6.15, dilution 2) is shown in Appendix 2.

The in vitro investigations of VAP stability reported here were performed in closed systems from which no significant withdrawals of each solution were made. In contrast, in the animal holding rooms, rats would be regularly drinking from the water bottles. The volume of water withdrawn during drinking would be replaced with an equal volume of air. This regular intake of air would be expected to increase the level of VAP breakdown still further in comparison to that observed in vitro.

Thus, experiments carried out in vitro to assess the stability of dilute solutions of VAP Type 100 in deionised water have demonstrated that VAP under these conditions is unstable and likely to break down significantly over 3 or 4 days, even in the presence of the antioxidants BHA and BHT and after oxygen has been initially removed from the water, as in the case of fresh glass distilled water. These experiments have explained why such a high level of vitamin A (3.0 pgRE/rat/day) was shown to be required when VAP Type 100 is administered in the drmking water for the long-term maintenance of vitamin A-deficient rats.

4 TRIAL V(B) AN INVESTIGATION OF THE EFFECT OF FLUCTUATING VAP