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GENERAL PLAN OF EXPERIMENT.

An extremely careful study of the whole problem involved suggested certain questions, the answer to which, if correctly given, might furnish positive evidence, or permit of the drawing of very close analogies.1

These questions, systematically arranged, are as follows:

I. Is the so-called preservative dissolved and absorbed by the body fluids?

II. If so absorbed, has the product any inhibiting or retarding influence on the digestive agents of the body?

III. Is the product in the strength wherein there is no appreciable retarding influence on the digestive agents in reality a preservative?

IV. What will be the effect of feeding to young animals for a considerable period in the food, such a quantity of a so-called preservative as will harmonize with questions II and III above?

V. What are the conditions, if any, that seem to require the employment of a preservative in food?

I.

Is the so-called preservative dissolved in and absorbed by the body fluids?

Through the courtesy of the Grand Rapids Veterinary College, in 1905 and 1906, the writer conducted experiments on healthy dogs, to ascertain the solubility in the digestive juices of the following so-called preservatives: Benzoates (sodium), Salicylates (salicylic acid), Borates (borax) and Formaldehyde.

In each instance (two dogs), sodium benzoate was apparently completely absorbed and taken into the system of the animal. No sodium benzoate nor benzoic acid was eliminated in the feces and no unchanged benzoic acid was found in the urine.2

The question as to whether benzoate of soda is absorbed in the system is of course an important one, and while because of its nature and its great solubility in water, it might be considered almost self evident that absorption would take place, yet an experiment of this nature could hardly with safety have started with such an assumption alone. There is a considerable difference in the solubility of sodium benzoate

'It is well to state at the very start the opinion prominent in the mind of the writer when the work was planned, that we are well aware that positive evidence of the injuriousness of any product may be established only by securing actual effects on human subjects. Regarding this, however, we will have more to say later. (See pages 30, 31.)

"This seems to be entirely in keeping with the results of other investigations on the fate of benzoic acid. It is entirely changed (in small quantities) to hippuric acid, whether eliminated completely in the urine or partially eliminated in the feces. (Parker & Lusk, Am. J. Physiol., 3-472.)

and that of benzoic acid, and inasmuch as sodium benzoate would be changed apparently to benzoic acid upon reaching the stomach, the question of absorbability, when administered in food, becomes a point for solution.

These observations throughout might profitably have been conducted by en-capsule administration, but here again another problem arises, to-wit, the possible effect of mass action, not only upon absorption of the sodium benzoate, but also upon cell function.

Wiley (Bulletin No. 84, Bureau of Chemistry, part 1, page 32) contends that the motor movement of the stomach would immediately guard against any possible mass effect, and there is no doubt that it would tend in this direction, but it seems doubtful if this contention will hold in its entirety, for it seems to us that this would mean a peristaltic effect not usually contemplated by most physiologists.

In studying drug action, the drug may be properly administered in capsule form, because physiological symptoms are desired, but we think a more logical method of studying a product used in food is to administer it in the food.

It is evident from this1 and the work of others (Loc. Cit.) that sodium benzoate is quite readily absorbed in the stomach and is eliminated quite largely in the urine, thereby demonstrating its entrance into the animal system proper. It should be borne in mind, then, that an effect of sodium benzoate a priori should be looked for, further than a possible mere retarding influence on the digestive enzymes.2

Certain evidence likewise that glycuronic acid contributes to the elimination of benzoic acid, thereby tending to suggest a reason why it has been impossible to account by analysis of the urine and feces for all of the benzoic acid, ingested, in the food. (Magnus-Levy, Bichhem. Zeitzch, 6-502, 1907.)

The above also serves to illustrate how easily cumulative effect may be erroneously inferred when the analyses are not accompanied by autopsy. (Author.) 2 Lehman (Chem. Ztg., 32-951) maintains that benzoic acid administered in the food is eliminated by man and by carnivora as hippuric acid so long as the quantity ingested is not too large. He claims in cases of benzoic acid ingestion that the synthesis of hippuric acid takes place in the kidneys. (An observation not without interest in connection with our investigations recorded later.)

Magnus-Levy (Bicohem. Zeitzch, 1907, 6 heft 5 and 6) obtained benzoic acid eliminated in the free state when great quantities were ingested, and attaches great significance to its combination with glycocoll, claiming thereby a withdrawal of this latter product from the system in an attempt to convert the benzoic acid into the more easily soluble hippuric acid, and thereby of a consequence, disturbing the metabolic balance. That this may be a by no means remote possibility is shown by his own data whereby in feeding considerable quantities to herbivorous animals (sheep), he did succeed in having free benzoic acid eliminated. However, injudicious feeding (over feeding) does not necessarily furnish data comparable to rational administration of a product, and does not of itself necessarily indicate what the fate of the product ingested may be under conditions nearer to normal.

Lewinski (Arch. Exp. Path. in Pharm., 51-397) maintains that very large quantities would be required to cause an elimination of free benzoic acid in the urine, and thus presents no counter evidence of its solubility and absorbability when taken in the food in small or moderate quantities.

So much data is at hand to show that sodium benzoate is absorbed and hence enters the system proper, that this point was considered established, and beyond the determinations above referred to, it was considered unnecessary to pursue this point further. (Author.)

Vaughan (Jour. of Proceedings of National Association State Dairy and Food Departments, 1904, page 200). Title, "The Effects of Aluminum and its Salts on the Health of Man;-Is Aluminum Hydrate Soluble in Digestive Juice?"

II.

If absorbed, has the product any inhibiting or retarding influence on the digestive agents of 'the body?

A. Action of Sodium Bensoate upon salivary digestion, in vitro.

EXPERIMENT I.

Into each of eight (8) Erlenmeyer flasks was put 100 cc. of saliva mixture (75 cc. water + 25 cc. fresh human saliva). There was then introduced varying amounts of Sodium Benzoate as follows:

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An equal quantity of cooked potato was then put into each of ten (10) test tubes, into the bottom of each of which had been blown a hole and which hole had been covered with a thick layer of glass wool on the inside of the test tube. The potato was placed on the glass wool and the tubes containing the potato were then lowered into the Erlenmeyer flasks containing the saliva mixture and all placed in the incubator over night.

In the morning, digestion had progressed in all of the tubes except No. 8 (2% benzoate) in which digestion had been completely inhibited. (Table A.)

There being in Expt. I, Table A, therefore no clew as to the rate of digestion in the different tubes, the experiment was repeated, varying the amount of enzymes acting."

EXPERIMENT II.

Quantities of Sodium Benzoate were exactly the same in this experiment as in Expt. I. The enzyme acting was contained in 10 cc. saliva diluted with 90 cc. water. Digestion again progressed over night. In the morning, digestion had proceeded as shown in Table B.

'It may be that a substance may retard or accelerate digestion at the start and yet not disturb its final equilibrium. See J. Biol. Chem., VI, No. 2, p. 138. "The greater the amount of enzyme acting, the less the inhibiting effect. See J. Biol. Chem., IV, 2 and 3, pp. 151-161. Loevenhart & Peirce.

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A third experiment, conducted as in Expt. II, gave the following:

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Pieces of glass tubing about three-fourths of an inch long were filled with cooked potato. These were put into test tubes containing saliva mixture as in Expt. II. Digestion here progressed two (2) days. (This time was necessary because of the small surface exposed to the enzyme.) At the end of this time, the digestion was stopped and the tubes preserved in glycerine. Results were obtained as follows:

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B. Action of Sodium Benzoate upon Gastric Digestion, in vitro.

EXPERIMENT V.

A series of test tubes, prepared as in the preceding experiments, but containing coagulated egg albumin, were placed in an artificial gastric juice containing varying percentages of Sodium Benzoate. Digestion

progressed 221⁄2 hours in the incubator at the end of which time the enzymes were rendered inactive by heating the flasks. Results as follows:

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Same as Expt. V. Digestion took place over night (12 hours).

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