Chester J. Farmer

Determination of reduced ascorbic acid in small amounts of blood.

We previously presented data as to the quantity of reduced ascorbic acid in blood plasma and showed its dependence upon the dietary supply of vitamin C, or of ascorbic acid administered as a medicament. 1 The data were obtained by deproteinizing plasma by means of tungstic acid and titrating immediately the reduced ascorbic acid present with 2:6 dichlorobenzenoneindophenol. In working with infants, and when making repeated observations on the same individual, it is desirable to have a method which requires a minimum of blood. We have, therefore, developed a micro-method requiring but 0.3 ml. blood, which may readily be secured from the finger of an adult, or the heel of an infant. In the guinea pig, blood is obtained by cardiac puncture, or sometimes by incision of the marginal vein of the ear. In the latter case, a special blood pipette is required. Collection of Blood. Collect approximately 0.3 ml. blood from a lancet wound, into a small phial (Fig. 1, B) containing sufficient powdered potassium oxalate to prevent coagulation. Stir immediately with a toothpick or small glass rod. The phial is made by sealing the end of a piece of glass tubing of approximately 10 mm. outside diameter, and, while hot, pressing it against a piece of iron to form a flat base. The tubing is cut off at about 15 mm. from the closed end. The cut end is then ground flat on a fine stone wheel and the outer wall ground to a taper for a distance of 5 mm. from the top. The grinding produces a thin edge which greatly facilitates the collection of blood from the heel of an infant. It is unnecessary to grind the side wall if the phial is used for finger blood only (which is permitted to drop directly into the phial as the finger is milked), or if Mood is transferred from a syringe. The phial is stoppered (Stopper A), then placed in a suitable holder (a cork with a recess cut in it) and lowered into the metal tube of a centrifuge. It is centrifuged for a few minutes in the usual manner.

Ascorbic Acid Content of Blood

While numerous papers have appeared dealing with the ascorbic acid content of various plant and animal tissues, and urine, only a few refer to the quantity present in the blood. Van Eekelen, Emmerie, Josephy and Wolff, 1 and Emmerie and Van Eekelen 2 deproteinize blood, blood plasma, or serum with trichloracetic acid, precipitate interfering substances, chiefly SH compounds, with mercuric acetate, then treat with H2S, which not only precipitates the mercury but also reduces that portion of ascorbic acid which in blood occurs in a reversibly oxidized state. The H2S is later removed by a stream of nitrogen, and the ascorbic acid estimated by titration with 2:6 dichlorobenzenoneindophenol. 3 Gabbe 4 claims that loss of ascorbic acid occurs if a solution of pure ascorbic acid is treated with H2S in the presence of mercuric acetate, and therefore, omits this step. Tauber and Kleiner 5 describe a method generally applicable to plant and animal tissues (and to blood) in which the essential features of deproteinization, removal of interfering substances, and H2S reduction are preserved. They estimate the ascorbic acid present either by titration or by its ability to reduce potassium ferricyanide, with subsequent development of Prussian-blue upon the addition of the ferric gum ghatti reagent of Folin and Malmros. 6 Our experience with several of these methods disclosed a number of difficulties. Some of the methods require considerable quantities of blood. Mercuric acetate solutions must not be over two weeks old, or filtrates containing colloidal sulphides may be obtained, particularly when the method is applied to urine. Any colloidal material in the filtrate makes it impossible to obtain a satisfactory endpoint during the titration with the dyestuff. The endpoint with trichloracetic acid filtrates is not sharp. In the present study, we are chiefly concerned with finding some method whereby the relative ascorbic acid level of blood in infants and children may be determined as an aid in studying subclinical scurvy and following its course under treatment.

Invalidation of plasma ascorbic acid values by use of potassium cyanide.

Conclusion For dependable ascorbic acid values, blood should be centrifuged, the plasma deproteinized, and the plasma-HPO3 filtrate titrated in immediate sequence after the blood is drawn. Whole blood which stands in a closed small phial, with a minimum air space, may be depended upon to give results of clinical value for 1/2 hour. The higher values obtained with bloods to which KCN has been added represent an enhancement due to the action of KCN upon the 2,6-dichlorophenolindophenol, and in no wise a more accurate determination of their ascorbic acid content. This is particularly true with blood of low ascorbic acid value. KCN does not prevent the loss of ascorbic acid from blood. It is a pleasure to acknowledge our indebtedness to Mrs. Jessie Maaske for technical assistance, and to Mr. Herman Chinn for checking several titrations.

Influence of arsenicals, bismuth and iron on the plasma ascorbic acid level.

Several reports indicate that the poisonous effects of a number of drugs like benzene, lead, 1 phenylcinchoninic acid, 2 and glycerol 3 and especially the arsenicals1 may be counteracted successfully by giving suitable doses of ascorbic acid. From examination of the urine Dainow 4 concluded that patients who showed symptoms of intolerance to arsenicals were in a state of hypovitaminosis C. By administration of ascorbic acid, these hypersensitive patients became able to tolerate neoarsphenamine. Other investigators 5 6 7 8 9 10 11 reporting similar observations, emphasize the fact that in certain hypersensitive cases ascorbic acid gave favorable results after other methods of detoxification such as the administration of glucose, invert sugar, and calcium or sodium thiosulfate had failed. After a suitable method for determining plasma ascorbic acid had been developed, studies were commenced in 1938 on syphilitic patients showing symptoms of intolerance to arsenicals. A more extended systematic study of this problem was recently made possible in connection with our Nutritional Survey of the syphilitic patients attending the Municipal Social Hygiene Clinic, Chicago.∗ It was noted early in the work that patients hypersensitive to neoarsphenamine in whom treatment had to be discontinued because of severe reactions‡ required exceedingly large oral doses of ascorbic acid† to bring their plasma levels up to optimal values (1.0 mg % or above). When showing severe symptoms of intolerance, a decline of the plasma level occurred in spite of the oral administration of ascorbic acid during treatment. It was frequently observed that a marked lowering of the plasma level followed the administration of neoarsphenamine in patients showing no intolerance to the drug (Fig. 1). When bisniuth was given in doses routinely used for aiitiluetic treatment, no appreciable effect was observed either on the plasma ascorbic acid or hemoglobin levels.

Determination of Ascorbic Acid in Feces. Its Excretion in Health and Disease

Conclusions 1. A method for the estimation of ascorbic acid in feces is described, and by its use, the fecal content of the normal individual on an adequate but unsupplemented diet is shown to average about 5 mg daily. 2. The plasma, urinary and fecal ascorbic acid contents have been followed in a normal individual after administration of varying amounts of 1-ascorbic acid by mouth. Large variations in the dietary intake were shown to affect the fecal excretion only slightly. 3. Patients suffering from certain gastrointestinal disorders excreted larger quantities of ascorbic acid in the feces than normal individuals.

Gastric Mucin Secretion in Gastro-duodenal Ulcerative Disease

It was shown 1 that commercial gastric mucin when subjected to peptic digestion and subsequent precipitation with alcohol at 70% concentration, yielded upon hydrolysis characteristic values for total nitrogen and total reducing substances. This suggested the use of a reduction method for the quantitation of mucin. Obviously before such a method can be applied to gastric juice, it is necessary to remove all other reducing substances, or to use a procedure for obtaining gastric juice not subject to their presence. The former procedure would be capable of wider application and work is in progress with this end in view. Our present interest is in the variations in mucin content of gastric secretion, when juice is obtained from the fasting stomach by means of an alcohol test meal under conditions excluding other reducing substances. In following the curve of mucin secretion, the stomach is emptied after a nights fast, basal secretory rate established, then 250 cc. of 7% ethyl alcohol is given and samples removed through a Rehfuss tube at 15 minute intervals for a period of 2 hours. Determinations of total and free acidity, and pepsin, are made. The mucin secretion is followed by the determination of total reducing substances according to the following method: The gastric sample (5 cc. if available) is diluted with an equal volume of 4 N H2SO4. It is then hydrolyzed in a boiling water bath for 2 ½ to 3 hours using an air condenser. After cooling, 2 cc. aliquot samples are pipetted into test tubes, the acidity neutralized to phenolphthalein with 1 N KOH or NaOH, and the reduction estimated by the Somogyi modification of the Shaffer-Hartmann method. 2 The results are expressed in milligrams of reducing substances (calculated as glucose) per cubic centimeter of gastric juice. To illustrate values found, the accompanying graphs are appended.

Influence of Catharsis and Diarrhea on Gastrointestinal Absorption of Ascorbic Acid in Infants

Summary Ascorbic acid is excreted in small amounts in the stools of the normal infant studied. Large amounts of orally administered ascorbic acid are excreted in the stools of infants following catharsis and during acute diarrhea. The increased fecal excretion of orally administered ascorbic acid during acute diarrhea in the infant points to its failure of absorption in the intestinal tract, and explains the low blood plasma values and low urinary excretion.

Streptomycin in experimental ocular infections.

Summary 1. The penetrability of streptomycin through the cornea may be increased by abrasion, inflammation, ion-transfer and wetting agents. 2. No local toxic effects were noted when saline solutions of streptomycin containing up to 10,000 μg per ml were used. With concentrations of 50,000 μg or as a dry powder, delayed healing occurred. 3. Intraocular injection, in amounts up to 1,000 μg of streptomycin in 0.1 ml saline were well tolerated. Smaller amounts (25 to 300 μg) were therapeutically effective up to 6 to 8 hours against a virulent strain of Str. pyogenes, though transient or negligible vitreous opacities occurred with these concentrations. 4. Experimental corneal ulcers produced by injection of B. pyocyaneus were prevented when treatment was started within 6 hours after inoculation by 3 applications at 2-hour intervals of a saline solution of streptomycin containing 10,000 μg per ml.

Studies on enzymatic digestion of gastric mucin.

Although it is generally assumed that mucin is digested in the intestinal tract, no systematic investigations on the enzymatic hydrolysis of mucin have been reported. We have, therefore, undertaken to study the action of enzymes upon mucin in vitro for the purpose of determining its possible manner of cleavage. The general procedure consisted in adding a known amount of enzyme to a mucin solution of known concentration at a pH well within the range of activity of the enzyme being tested. Controls consisted of mucin alone and enzyme alone at approximately the same pH. At intervals, usually 2 days, samples were removed and analyzed, until no further breakdown beyond that occurring in the controls could be detected. Proteolysis was followed by the nitrous acid method of Van Slyke. 1 Cleavage of the carbohydrate portion of the molecule was tested for by determining the presence or absence of reducing substances, using the Somogyi-Shaffer-Hartmann method. 2 The extent of cleavage was also determined by the decrease in the amount of mucin precipitable in 70% alcohol, and subsequent analysis of the precipitated material. The activity of all enzymes used was demonstrated by their hydrolysis of suitable substrates. In those cases in which digestion of mucin was observed, a second portion of enzyme was subsequently added to preclude the possibility that cessation of hydrolysis was due to depletion or inactivation of the enzyme. We have previously reported the isolation of a purified mucin from commercial preparations by a process of peptic digestion followed by 70% alcohol precipitation. 3 Since peptic digestion is used in the preparation of commercial mucin, the additional peptic digestion was employed for the sole purpose of rendering alcohol soluble any extraneous protein material present as a result of improper manufacture, or subsequently added as a diluent.

Influence of Hyperpyrexia on Ascorbic Acid Concentration in the Blood

Conclusions A study of 77 observations made on 17 patients showed no significant changes in the ascorbic acid concentration as the result of elevating the body temperature to 104°F and maintaining it for 4 hours, using a combination of air conditioned cabinet and indnctotherm.