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By Rodolfo Paoletti, Dr. David Kritchevsky

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1 N bromine (1 ml of Br2 in 390 ml of 2 % KBr) or in saturated bromine water for 1 minute. (c) Wash in water, rinse in 1 % sodium bisulfite for 5 minutes, and wash seven times in distilled water. (d) Treat with 1 % silver nitrate in N nitric acid for 18 hours. (e) Wash seven times in distilled water. (f ) Develop for 10 minutes in Kodak Dektol developer (or equivalent) diluted with water (1:1 v/v). (g) Wash well in water and mount in glycerine jelly. Some unsaturated lipids are stained brown-black (see Section III,B,3).

V) Lipids that normally stain black with OTAN appear orangebrown if they are only stained for a few seconds with O s 0 4 before treating them with α-naphthylamine. (vi) The red or orange-brown OTAN reaction may indicate a low density of unsaturated (ethylene) groups in the lipid, whereas the black reaction may indicate an abundance of such ethylenes. These criticisms can be countered by the following arguments: (i) Acetone essentially extinguishes the staining of atherosclerotic plaques with all phospholipid methods tested (see Section II,C,3); the explanation for this general anomaly remains obscure and requires further investigation.

Albumin and globulin, however, showed an outward-inward gradient in normal and slightly atheromatous vessels, indicative of diffusion of plasma proteins from outside-inward. , was inside-outward), suggestive of a direct leak of plasma proteins from the lumen into established severe lesions. These preliminary results are not consistent with the view that plasma proteins (in particular ß-lipoprotein) transport cholesterol across the normal and slightly atheromatous arterial wall. It seems more likely that local synthesis of phospholipid or protein within the arterial wall provides the vehicle for trans-mural cholesterol transport.

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