Thursday, May 8, 2014

SFM Dietary Protocols: Depleting Retinol


Our project to replace Rhodopsin with Porphyropsin in the human eye in order to affect a shift of the range of light that is perceivable can easily be summed up in three steps: deplete stores of vitamin A, administer A2, and measure the changes that occur. While these summations are valid, they don't do justice to the intricacies involved in each step. This section discusses the steps taken to deplete retinol levels.
To begin, it's important to review the three Retinoids that will be discussed: Retinol, Dehydroretinol, and Retinoic Acid. Retinol is the primary form of Vitamin A that's stored in the liver, circulated in the blood, and used in it's retinaldehyde form in the eyes as a visual pigment. Depleting this retinol is essential for the success of our project as the Opsin proteins preferentially bind to Retinol.
Depleting the retinol facilitates opsin molecules binding instead to dehydroretinol – a retinoid found in the eyes of amphibians and freshwater fish which perceive a considerably altered visual range. Finally, we have Retinoic Acid which is a retinoid formed from the degradation of Retinol and which serves a vast number of functions at the cellular level but doesn't play a role in the visual cycle.
The first and most overt step in depleting Retinol is all subjects maintaining a Vitamin A deficient diet. The term vitamin A refers to the retinoids already discussed as well as other precursor molecules such as Beta-Carotene. This is a more difficult step than one might expect. Avoiding all foods containing Vitamin A is easy; however this diet is to be maintained for a 60-90 day period and a diet free of retinoids, but otherwise nutritionally complete is nearly impossible. After a number of attempts to design such a diet, we decided to instead forgo “food” altogether and to fulfill our needs with a nutrition shake produced by Manufacturer “Soylent.” SFM purchased a custom run of Soylent containing all macro and micro-nutrients needed to prevent a physiological deficit except Retinoids. Analysis of ingredients led to the further modification of removal of Maltodextrin in order to increase the protein to carbohydrate ratio.
A VAD (Vitamin A Deficient) diet is a start, but two glaring problems remain. One is that a VAD diet comes with significant physiological ramifications. The other issue is that retention of Retinol in livers is highly variable. Humans subjected to a VAD diet at times develop night blindness in as little as two weeks, while other can remain in VAD for up to two years before experiencing mal effect.
The first symptom of a subject with VAD after depletion of retinol stores is night-blindness4. This night-blindness is related to degradation of the visual pigment Rhodopsin. Rhodopsin is constructed from an opsin protein and Retinol and thus in VAD can't form. The next symptom is the sharp decrease in the production of the protein opsin itself. The third symptom is deterioration of outer segments of rod cells themselves. It's at this time that other more overt and life threatening symptoms begin, including loss of weight, postural imbalance, respiratory disturbance, corneal opacities, and eventually death.
Fortunately, the systemic changes such as death are completely preventable with the administration of Retinoic Acid4. Under normal physiological circumstances, retinol circulates in the blood and is delivered to the cells of the body which then degrade it into Retinoic Acid (RA). Exogenous RA is able to completely replace the role of Retinol in the human body except for its role in the visual cycle and in spermatogenesis6. Prevention of death may seem an obvious reason to administer RA while maintaining a VAD diet; however, even with RA supplementation depletion of retinol leads to first night-blindness, then a decrease in Opsin proteins, and finally the degradation of the cellular layers themselves. It's fortunate that except in cases of particularly extended VAD these visual disturbances are reversible.
Research performed by G. Wald (1960) demonstrates that Rats kept on a VAD diet with RA supplementation for up to ten weeks experienced a rapid return of visual acuity following administration of Retinol. Rats maintained in VAD with RA for 25 weeks demonstrated first a period of rapid visual improvement followed with an extended period of slow improvement back to baseline, representing the return of opsin synthesis and structural repair of visual cells. Rats kept on a VAD diet with RA for longer than six months never returned completely to baseline. Retinoic Acid certainly prevents the worst symptoms of VAD, but a much better alternative is available: Dehydroretinol.
The physiological functions performed by Retinol related to reproduction, growth, and vision can all be fulfilled by dehydroretinol5,6. Furthermore, the dehydroretinol is either utilized as is, or is degraded to retinoic acid as there is no evidence that dehydroretinol can be reduce to retinol. It doesn't become retinol, but rather replaces it in the eyes to form Porphyropsin – the very visual pigment that allows freshwater fish such an extended visual spectrum. Were dehydroretinol inexpensive and widely available, one could perform this project in its entirety using nothing more than a VAD diet and dehydroretinol over a particularly extended period of time. The problem that arises is that liver storage of retinol is notoriously tenacious. As long as the basic metabolic functions are provided for by exogenous dehydroretinol, the liver will mete out only the miserly quantities of retinol needed to maintain normal vision1. The process could take years. While Retinoic Acid is limited in it's ability to prevent visual degradation, it still fulfills a vital role in our process to facilitate a change in vision.
A 1974 study performed by M. Rao found that administration of RA leads to a rapid decrease in circulating Retinol. The underlying mechanism was elucidated by Keilson in “Effects of Retinoic Acid on the Mobilization of Vitamin A from the Liver in Rats.” This study showed administration of RA spares the release of retinol from the liver causing the decrease in serum values. It can take years to deplete liver stores of retinol with VAD diet alone, but by concurrently administering RA with the dehydroretinol, we can sequester the retinol in the liver facilitating a much greater relative availability of dehydroretinol to retinol. Retinoic acid has also been shown to play another valuable role prior to administration of dehydroretinol.
G. Wald made note of a rather unusual finding regarding RA, but provided no attempt at explanation: Rats whose VAD diets were supplemented with RA experienced night-blindness far more rapidly that rats on a VAD diet alone. This finding was further explored in the 1974 study, “Induction of Rapid, Synchronous Vitamin A Deficiency in the Rat.” Cycling between periods of VAD dieting alone, and with periods of RA administration were found the optimal method to deplete retinol while maintaining subjects health. This adds a final modification to our supplementation regimen. The study found the optimal period lengths of RA supplementation to total retinoid deprivation to to be 18:10. It's likely that cycling prevents the Retinol sequestering effect in the liver of continuous RA administration, while also preventing a decrease in metabolic processes using Retinol that would otherwise occur in order to conserve Retinol with a VAD diet alone. Seeing as though we're performing this procedure on humans and more specifically upon ourselves, we decided upon a much more conservative 9:5 ratio.
Our methodology for Retinol depletion is as follows:
Initial VAD Period
Days 01-05



RA Cycle 1
Days 06-13



Deprivation Period 1
Days 14-19



RA Cycle 2
Days 20-29



Deprivation Period 2
Days 30-35



RA Cycle 3
Days 36-45



Deprivation Period 3
Days 46-51




At day 51, the dehydroretinol phase of the study begins which will be addressed in a further post. This regimen allows for 3 completed cycles alternating between complete retinoid deprivation and retinoic acid administration. Alternatively, if overt signs of VAD are assessed then the dehydroretinol administration is to begin immediately for all participants. While a longer and more severe system of Retinol depletion prior to beginning the dehydroretinol administration is possible, this regimen is considered a conservative and safe compromise.

Citations
1. Keilson, B., Underwood, B., & Loerch, . U. Effects of Retinoic Acid on the Mobilization of Vitamin A from the Liver in Rats. J Nutr, 109, 787-95.

2. Rao, M. Et All. Vitamin A metabolism under nutritional stress.1974 Int. J. Vitam.
Nutr. Res. 44, 151-157
(Administration of RA causes an immediate serum decrease of Vit A )

3. Lamb, A,. Olson, J. Et All. Induction of Rapid, Synchronous Vitamin A Deficiency in the Rat. 1974, J. Nutr. 104: 1140-1148

4. Wald, G. The Biological Function of Vitamin A Acid. 1960 Proc Natl Acad Sci U S A. May 1960; 46(5): 587–608.

5. Bridges, C., Hollyfield, J., The visual pigment and vitamin A of Xenopus laevis embryos, larvae and adults. 1977 Experimental Eye Research, Volume 24, 1: 7-13

6. Howell, J., Reproduction and Vision in Rats Maintained on A retinol Free Diet containing 3-Dehydroretinol (Vitamin A,2) 1966, Br. J. Nutr 21, 373






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