The need to have quantitative information about the amount of double bonds present in the organism or in its lipoacids has led to a method of analysis based on the fact that molecular breakdown or fission will furnish characteristic components. With fission occurring at the level of the double bonds, the fraction corresponding to a conjugated double bond will appear as oxalic acid. The problem was to obtain this fission with a carboxyl corresponding to each carbon and without having artificially induced displacements of the double bond which is a frequent result of treatment.

We employed the following technique. Fatty acids from an organism or any other preparation were neutralized with the exact amount of sodium carbonate necessary. This amount was established through the neutralization index of the substances to be treated. After sufficient dilution, an excess of sodium carbonate was added with the aim of obtaining an alkaline medium. After bringing the solution to 4°C potassium permanganate was added until further discoloration of the permanganate stopped, after which 20% more of the amount already used was added. The mixture was kept refrigerated at 4°C for 16 hours, after which the excess of permanganate was reduced by sodium bisulfide. The liquid obtained was filtered and the precipitate washed. The liquid was extracted first with ether to eliminate the higher fatty acids, after which it was submitted to distillation in order to eliminate the volatile fatty acids. In the remaining part, the oxalic acid was precipitated with calcium chloride. From the precipitate, the part corresponding to calcium malonate was separated from calcium oxalate, by using the difference in solubility at the boiling temperature. The oxalic acid was then titrated in the usual manner. The amount of oxalic acid divided by the quantity of lipoacids used represents the oxalic index of the preparation. Carlos Huesca Mejia and Daisy Franco have widely studied the changes of this oxalic acid index in our laboratories.

Pure nonconjugated fatty acids treated in this manner yield no oxalic acid. When linoleic acid is conjugated (e.g. by treating with KOH in ethylene glycol) oxalic acid is found in the fission products in amounts that gradually change as the treatment continues. (Table XXXIV)

Table XXXIV

The Quantity of Oxalic Acid Present After Oxidative Fission and Iodine Number of Samples of Linoleic Acid Conjugated for Different Periods of Time. (Linoleic Acid Mixed With Equal Quantities of KOH: Dissolved 5% In Ethylene Glycol; Continuous Treatment In Reflux.)

Time

Oxalic Acid mg gm of Fatty Acid

Iodine Number

Before treatment

0

180

After 30 minutes

117

119.8

" 1 hour

205

115.1

" 2 hours

114.2

94

" 4 hours

119.4

96

" 8 hours

99.9

91

" 12 hours

92

86

" 24 hours

85

81.7

" 36 hours

80

76

" 48 hours

77.3

76.5

" 144 hours

40.8

57.3

The quantitative relationship between known proportions of conjugated fatty acids and the oxalic acid obtained through their oxidative fission has been studied.

Autolytic changes have been found to influence the nature of the fatty acids extracted from tissues. Studies have shown that formalin fixation does not significantly change the fatty acids present. (Table XXXV)

It has been noted that relatively stronger methods of conjugation, utilizing KOH at higher concentration and higher temperature, are necessary to conjugate the fatty acids of normal tissues, than are needed for the fatty acids extracted from pathological tissues (burn, shock, adrenalectomy, tumor necrosis) which can readily be conjugated by much milder procedures.

Table XXXV

Effect of Formalin Upon the Quantity of Oxalic Acid Produced By Oxidative Fission of A Mixture of Conjugated Fatty Acids. (5 cc Aliquots of A Mixture of Conjugated Fatty Acids Were Mixed In A Stoppered Cylinder With 10 cc. of A 20% Formol Solution, the Cylinder Was Shaken and Samples Were Removed At Frequent Intervals For Determination of Oxalic Acid.)

Time

Mgm Oxalic Acid/gm Fatty Acids

Before mixing

121.7

After 1 hour

121.8

" 24 hours

120.5

" 48 hours

120.9

" 72 hours

118.9

" 144 hours

121.9

Chapter 10, Note 2. Irradiation And Oxalic Index

Forty male albino rats separated in groups of 10 were irradiated with nonfiltered X rays from a 200,000 v. machine, receiving in one session a dose of 1500 r. which is considered a lethal dose. Similar experiments were repeated several times, some with the radiation dose obtained from radioactive cobalt. The four groups of animals were first mixed together and then separated in four big cages. The animals and controls were kept on Purina Chow and water as libitum. Two of the controls, two of the treated animals, and any others approaching death were sacrificed daily.

Each dead animal was saponified separately. The total amount of acid lipids extracted was analyzed for conjugated fatty acids, using the method indicated previously in which the amount of oxalic acid which appears as the result of oxidative fission is measured. The values were expressed in oxalic index, which corresponds to the amount of oxalic acid in milligrams per gram of fatty acids. Figure 85 shows the results of two such experiments.

Values found for normals were zero or less than 0.5, but a constant increase of the oxalic index was noted after irradiation. While irregular values were still seen during the first three days, all were above 3 on the fourth day and continued to increase constantly afterward. Death occurred when the oxalic index reached a critical point which was found to correspond to 14-17 mgr. of oxalic acid per gram of the fatty acids of the entire body.

Chapter 10, Note 3. Oxalic Index In Sublethal Irradiation

We studied the changes in oxalic acid in animals treated with an amount of radiation below the lethal doses. The oxalic acid rose after treatment with values much lower than those seen when lethal doses were used. Three groups of ten animals each were radiated with 600 r., and every few days two of the animals were sacrificed along with two of the controls. Figure 91 shows the values obtained for these animals. It could be seen that although indices of 6 and 7 were found, these never reached the critical point of 14, and that the indices went down in an irregular fashion. While some animals had values of around 4 and 5 after the tenth day, others had high values at the same time.

The administration of polyunsaturated conjugated fatty acids (1-2 cc. daily of a 5% oily solution of conjugated cod liver oil fatty acid) induced death in a high proportion (16/20) of animals irradiated with otherwise nonlethal doses such as 800 r. In these animals the oxalic index was high, often showing values above 17.