Quantitative histochemistry of nicotinamide adenine nucleotides in retina of monkey and rabbit.

Abstract

Abstract—The levels of NADP+, NADPH, NAD+ and NADH were measured in the different layers of retinas from rabbit and monkey. Samples (0.1 μg) were dissected from frozen‐dried sections. The sum of oxidized and reduced forms was obtained by analysis of samples diluted several thousand fold in 0.02 n‐NaOH at 0°. The reduced forms were measured by analysis of the same alkaline preparation after heating to destroy NADP+ and NAD+. All assays were made at 1:100,000 tissue dilution by enzymic cycling, which is capable of measuring 10−14 moles of nucleotides.Profiles of nicotinamide adenine nucleotide levels werecomparable in monkey and rabbit. Both total NADP and NAD were lowest in the outer segments of the retina and highest in the inner layers. NADP of the outer layers (1‐2b) was oxidized to a high degree. This was particularly striking for layer 2b, which is rich in mitochondria. In the inner layers the fraction of NADPH rose to 0.7 of the total NADP. NAD on the contrary was highly oxidized in all ten layers of the retina.Three aspects of these results seem significant: (1) The profile for NADP was not related to the distribution of any of four major NADP‐requiring dehydrogenases or their sum; (2) the ratio of total NADP/NADPH in the mitochondrial layer was much higher than expected from studies with isolated mitochondria; and (3) the amount of total NADP was surprisingly high in non‐mitochondrial layers.