Reply to “Letter to the editor: ‘Comments on retinal metabolic state in P23H and normal retinas’”

Abstract

REPLY: B. Winkler questions the validity of some of our conclusions in relation to a mutant model of retinitis pigmentosa (Proline-23-Histidine rat) published in a recent issue (1). He bases his objections on three issues: 1) Lactate dehydrogenase activity in the P23H does not appear to be elevated during the early critical stages "of the 8 days that were sampled between P1 and P21, there were only small (Ͻ5%), statistically insignificant differences in LDH activity between SD and mutant retinas". 2) Calculating the total ATP content appears to be greatly elevated. 3) ATP location within the vertebrate retina suggests altered ATP levels may be irrelevant.We have addressed the three issues below. There is no change in our conclusions that LDH activity and ATP content are both elevated early in development of the P23H rat retina, as is Na ϩ -K ϩ -ATPase activity. Are LDH Levels Significantly Elevated in the P23H Rat Retina?LDH activity is a signature of the metabolic status of the photoreceptors, particularly after degeneration (1, 4). We have previously shown that in the mouse model of retinitis pigmentosa (rd/rd or rd1 mouse), there is a significant increase in LDH activity during the early critical ages (1) followed by a decrease when many photoreceptors have degenerated: similar to what occurs in the P23H rat. It is not clear how Winkler concluded that there are "statistically insignificant differences in LDH activity between SD and mutant retina", when no statistical tests were cited for such a statement.ANOVA is a parametric test that analyzes the mean across groups reducing the chances of committing a type I error. The averaged percentage change across ages is small but significant (P1 ϭ 8%, P4 ϭ Ϫ3%, P6 ϭ 2%, P10 ϭ 18%, P12 ϭ 3%, P15 ϭ 4%, P17 ϭ 13%, P21 ϭ 22%). Statistical analysis using two-way ANOVA showed that LDH activity between P1 and P21 was significantly higher in the P23H-3 than in SD retinas (P Ͻ 0.01) (2).We have now also applied a nonparametric binomial test. The binomial test does not take into account the strength of the changes but calculate the probability that the increase-decrease is due to chance (probability 0.5). Across P1-P21 ages we observed that in 38 of 49 trials, LDH levels were increased in the P23H retina when compared with the SD retina. The two-tail P value is 0.0001. We reject the null hypothesis that LDH levels are equal in the two populations. Total Retinal ATP Content Appears to be Greatly ElevatedIn referring to the concentration of ATP in the retina, Winkler applies the correct analysis but assumes that our data can be directly transformed. We express our values in millimoles per gram protein secondary to perchloric acid extraction Address for reprint requests and other correspondence: M. Kalloniatis, ). Fig. 1. Total ATP and phosphocreatine (PCr) content in the developing SD and P23H retina expressed in mM per retina.