Dogs with
prcd have abnormalities in long-chain
polyunsaturated fatty acid metabolism, with reduced DHA levels
(approximately 20%–25%) in plasma and ROS and slightly elevated
levels in the liver, compared to unaffected dogs.
1 2 The
mechanism for the reduction in ROS DHA levels in
prcd-affected animals is not clear. It was initially thought
that synthesis of DHA in the liver and/or transport to the target
tissues could be affected. We tested this hypothesis previously and
tried to bypass the possible defect in DHA production in the liver by
providing preformed DHA to affected and normal dogs for up to 5
months.
2 We found that supplementation caused a rapid and
sustained elevation in plasma DHA levels in both groups, but had no
effect on the progression of the disease in
prcd-affected
animals. The
prcd-affected dogs fed a DHA-enriched diet
still had significantly lower DHA levels in ROS compared to control
dogs.
2 In dogs supplemented with DHA for up to 24 days,
levels of DHA were lower in ROS and higher in the liver from
prcd-affected dogs. These studies indicate that the lower
content of ROS DHA in
prcd-affected dogs is not the result
of a dietary deficiency of DHA and its precursor fatty acids. Besides a
possible problem in packaging DHA into lipoproteins in the
liver,
6 these results suggest the possibility that the
problem in
prcd-affected animals could be in DHA trafficking
between the blood and the retina, possibly involving passage through
RPE cells.
In this study, we investigated a possible defect in RPE of
prcd-affected dogs in the synthesis and release of DHA to
the extracellular space, by comparing these two activities in the RPE
of
prcd-affected and normal animals. This is important
because RPE cells could use 22:5n-3, which is slightly elevated in the
plasma of affected animals,
1 to synthesize DHA to
compensate for the reduced DHA levels in ROS of
prcd-affected animals. RPE cells of frogs have been shown to
readily synthesize DHA from its precursors.
7 Also, because
RPE cells are the major nutrient supplier for the photoreceptors and
play central roles in recycling of DHA from daily shed ROS tips back to
photoreceptors,
8 9 it is conceivable that alterations in
DHA metabolism in the RPE could affect photoreceptor DHA content. We
found that RPE cells from both
prcd-affected and normal dogs
can elongate 20:5n-3 to 22:5n-3 and further convert 22:5n-3 to 22:6n-3
through Sprecher’s Δ4-desaturase independent pathway
10 (22:5n-3 → 24:5n-3 → 24:6n-3 → 22:6n-3). Rates for DHA synthesis
appeared only slightly higher for affected RPE compared to normal
cells. Thus, in agreement with our previous in vivo
studies,
11 there is no defect in DHA synthesis in the RPE
of
prcd-affected dogs. Furthermore, under our experimental
conditions, RPE cells from
prcd-affected animals showed no
apparent defect in releasing DHA, inasmuch as the level of DHA in the
culture media from affected cells was clearly not lower than that from
normal cells.
It is noteworthy that the labeling pattern in the culture media did not
simply reflect that of the cells. When the distribution of
radioactivity was compared, clearly more DHA and less 22:5n-3 was found
in the media than in the cells. Thus, dog RPE cells appeared to favor
the release of DHA over 22:5n-3, although both are similar in
structure, with 22:5n-3 having one less
cis double bond.
Evidently the types of fatty acids in the media are not the result of
equilibration, but rather of cellular regulation, which also has been
demonstrated in retinal and cerebral microvascular endothelial
cells.
12 13
In light of our earlier
2 11 14 and present studies on
prcd-affected dogs, it is possible that lower ROS DHA levels
could be due to cellular upregulation of DHA catabolism and/or
downregulation of DHA incorporation into membranes to reduce
disease-originated metabolic (perhaps oxidative) stress. DHA provides
optimal lipid environment for rhodopsin and probably other membrane
proteins in photoreceptors and thus is essential for optimal
photoreceptor function. On the other hand, DHA is highly unsaturated
(six double bonds) and easily peroxidized, especially in the
photoreceptor cells, where high levels of oxygen and unsaturated fatty
acid content and light exposure provide the ideal environment for lipid
peroxidation. Toxic factors from lipid hydroperoxides derived from DHA
could affect photoreceptor enzyme activities and damage photoreceptor
membranes, as has been reported for light damage in albino
rats.
15 16 17 Biological adaptation, such as shortened ROS
length, reduced levels of rhodopsin and DHA-containing glycerolipids,
and increased antioxidants vitamin E and vitamin C and
glutathione-dependent enzyme activities, as well as neurotrophic
factors, in response to light stress has been shown in the retinas of
rats raised in bright cyclic light compared to controls raised in dim
cyclic light.
16 17 18 19 Similarly, oxidative stress achieved
through the stimulation of endogenous oxidant generation in human
spermatozoa can cause DNA fragmentation and loss in their capacities
for movement and oocyte fusion.
20 Therefore, it is
possible that reduction in DHA levels in retinas and plasma could be
part of a biological adaptation to metabolic stress, possibly
oxidative, caused by different mutations in retinitis pigmentosa. We
currently are testing the hypothesis that mutations in a number of
genes encoding retina-specific proteins can cause lower DHA phenotypes.