There is an increasing interest in the daily urinary excretion of vitamin C (cevitamic or ascorbic acid) since many infections, if not most, are accompanied by considerable diminution in such excretion. It would seem that under such circumstances the patient should have the optimum vitamin C level in his body maintained by proper diet or dietary supplement. Otherwise something approaching subclinical scurvy may conceivably become an annoying complication.
It is unfortunate that the oxidation losses in twenty-four-hour specimens as collected in actual practice are often so great as to mislead the physician in his diagnosis.
Using the Harris and Ray 1 method of titration against a fresh solution of the dye, 2,6 dichlorophenolindophenol, we determined the cevitamic acid content of fresh urine and of the same specimen after twenty-four hours. For this particular experiment the urine was kept in a clear glass bottle, open to the air. The loss (by action of light and oxygen) was 100 per cent. King and others attempted to preserve the vitamin by the aid of acetic acid (8 per cent of the total volume) and metaphosphoric acid (2 per cent of the total), or metaphosphoric alone, and gained some degree of success by so doing. We tested the inhibiting effect of acetic acid (10 per cent of total volume, as effective as the two acids together) and found a loss of 46 per cent of the vitamin in twenty-four hours. Even in twelve hours a 32 per cent loss was observed. To be sure, this was in warm summer weather with greater speed of oxidation than would be observed at lower temperatures. The open bottle test was severe. Yet air in partly filled bottles must be considered.
It is not permissible to get around this loss by analyzing a single specimen of freshly voided urine because the vitamin C content of urine voided at different times in the day or night varies greatly. If a considerable amount of the pure vitamin is given to a healthy person, saturated with the vitamin, urine excreted within an hour or two may contain the greater part of the extra dose. Evidently the safer course is to collect and preserve properly the four or five specimens usually voided during twenty-four hours. The patient must be warned that this does not mean beginning at 7:00 A.M. and finishing with another 7:00 A.M. specimen. He should discard either the first or the last 7:00 A.M. urine.
If the usual twenty-four-hour collections were in the physician’s hands six hours after the end of the period and he was free to make an analysis at once (often impossible), the average loss in warm weather might approximate 40 per cent of the vitamin originally present in the total excretion.
We decided to improve the methods of preserving urine during the collection period and the hours of delay in analysis.
Immediate chilling of each specimen was a distinct help, but impracticable. In whose refrigerator would bottles of urine be welcomed?
Exclusion of light by use of brown bottles proved to be a help in protecting the vitamin, but this was not enough.
A bottle filled to the cork was preferable to one containing a considerable air space, but collections are not usually of any desired volume. Partly filled bottles will be the rule, not the exception. Furthermore, in the act of voiding urine into a bottle some air is dissolved.
Common corks are porous enough to admit air on several hours’ standing, so we finally selected brown bottles closed with screw caps of plastic, not metal. Rusty metal caps catalyze oxidation of vitamin C.
Exclusion of air by carbon dioxide was successfully achieved by dropping small fragments of dry ice, solid carbon dioxide, into each bottle as urine was voided. The effect was excellent due to chilling and exclusion of air, but sometimes too much pressure was set up after closing the bottle. The inconvenience of securing the dry ice is a strong factor against its use.
Finally we adopted the plan of adding a slightly rounded teaspoonful (about 3 gm.) of precipitated calcium carbonate (a fine dry powder) to the bottle of fresh. urine into which 50 c.c. of glacial acetic acid had already been measured. After about half a minute of frothing to permit escape of air and carbon dioxide, the cap was screwed very tight. The escaping carbon dioxide drove out dissolved air as well as free air in the bottle. The resulting success suggested a final precaution: the extra addition of a fragment of marble, the size of one or two grains of corn (about 2 gm.). The quick action of the powdered calcium carbonate on the acetic acid was effective before capping the bottle while the slow action of the fragment of marble continued for several hours, setting up a small positive pressure of the gas, very helpful if the cap or stopper was not airtight.
The astonishing gain in accuracy due to this simple method of collection is shown in Table I.
| TABLE I Loss or Vitamin C in Urine Under Various Conditions or Collection (Fresh Urine in 500 c.c. bottle, only partly filled, warm weather) |
|||||
|---|---|---|---|---|---|
| BOTTLE | ACID | CaCO2 | COVER | TIME | LOSS OF VITAMIN IN PER CENT |
|
|
|||||
| Clear glass | None | None | Open | 24 hr. | 100 |
| Clear glass | 10% acetic acid | None | Open | 24 hr. | 46 |
| Clear glass | 10% acetic acid | Fragments | Screw cap too quickly closed | 24 hr. | 20 |
| Brown | 10% acetic acid | Fragments | Screw cap too quickly closed | 24 hr. | 3 |
| Clear | 10% acetic acid | Powder and fragments | Capped after half minute | 24 hr. | None |
| Brown | 10% acetic acid | Powder and fragments | Capped after half minute | 48 hr. | None |
Using the chemist’s Bunsen valve, which permits gases to pass out from the bottle but allows no air to enter, we found no measurable loss in forty-eight hours. This device, with small lumps of dry ice, is perfect but rarely usable for the average patient.
Suitable 500 c.c. amber bottles with screw caps of plastic (no metal) may be bought through dealers who in turn may buy from the Libby Owens Illinois Glass Co., of Toledo. Two types of bottles, the narrow and wide mouth, are needed.
Mold A-6338, 500 c.c. (16 oz.) narrow mouth, round, amber glass, 28 mm. 0.1 G-419 finish. Black molded caps—18 mm.—419 with cork and vinylite liners. Mold A-5160, 500 c.c., wide mouth, amber glass, 53-400 finish. Black molded caps—53 mm.—400. Pulp and vinylite liners.
To simplify collections the 3 gm. of dry precipitated calcium carbonate powder and a 2 gm. fragment of marble should be placed in a sealed envelope attached to the bottle by band or cork. The patient should be instructed to allow frothing to continue fully half a minute (to insure displacement of air by carbon dioxide) before tightly closing the bottle. Bed patients should void into some other vessel due to the possibility of acid burns.
If the foregoing directions are followed, urine may be preserved forty-eight hours without appreciable loss of vitamin C. Otherwise half or all of the vitamin may be lost in twenty-four to thirty-two hours.
Mix rapidly contents of all the bottles representing twenty-four hours collections, measure with a 500 c.c. graduate, and pour a small sample quickly into a 2 c.c. burette, graduated in hundredths, keeping the stopcock open until the liquid just reaches the stopcock. Wait nearly one minute or until the carbon dioxide bubbles rise and escape before again opening the stopcock and filling the burette to the tip. Run out liquid until the upper level is at the zero or starting point on the scale. Discard any urine escaping from the tip at this stage.
Titrate the urine into a 15 c.c. conical centrifuge tube containing 0.05 c.c. of a standard solution of the reducible dye, 2,6 dichlorophenolindophenol until the pink color following the blue of the dye weakens and becomes barely noticeable, remaining so for thirty seconds. It is helpful in this color comparison to hold a similar centrifuge tube of urine, diluted to the same extent, beside the sample in titration. The end point is secured when only a very slight difference in tint appears (against a white paper background).
Hofmann-La Roche, of Nutley, N. J., sell conveniently standardized dye tablets, each one sufficient to react with 1 mg. of vitamin C.
To make a standard solution, place one tablet in any very small bottle (or 20 c.c. flask) and add one drop less than 2 c.c. of distilled water from a 2 c.c. pipette. This allows for the slight increase in volume of the solution. Allow to stand a few hours to secure complete solution of the dye.
A daylight lamp is an aid in accurate color readings. Not more than two minutes’ time is allowable for a single titration.
Calculation. — For example, suppose that it required 1.4 c.c. of urine of a 500 c.c. specimen to decolorize 0.05 c.c. of the standard, then
1.4 c.c. urine should contain 0.025 mg. of vitamin C.
The 0.05 c.c. of dye solution is 1/40 of the 2 c.c. which was found to be equivalent to 1 mg. of vitamin C.
Therefore, 1 c.c. urine contains 0.025÷1.4 mg.= 0.01785
mg.
and 500 c.c. contains 500 x 0.01785 mg. = 8.9+ mg. vitamin C.
Other reducing substances often present during illness may introduce some error, but efforts to remove them introduce other errors.
The materials necessary for titration include: Microburette, 6-414-J, 2 c.c. graduated in 0.01 c.c. divisions, funnel top. Fischer Scientific Co., Pittsburgh.
Pipette, 13-674, capacity 0.1 c.c., graduated in 0.01 c.c. divisions, Fischer Scientific Co. Pipette, 2 c.c. Mixing bottle, 2 liters. Burette stand, small size. Graduate, 500 c.c., to measure total urine volume. Beaker, 50-100 c.c. Dye tablets of 2,6 dichlorophenolindophenol, Hoffmann-La Roche, Nutley, N. J. Glacial acetic acid (50 c.c. for each specimen). Marble, fragments of 2 gm. each, size of grain of corn. Calcium carbonate powder (precipitated).
Hawk: Practical Physiological Chemistry, ed. 11, Philadelphia, 1937, P. Blakiston’s Son & Co.; Lancet 228: 71, 1935.
From Journal of Laboratory and Clinical Medicine, Volume 24, 1939, pp. 1293-1296.
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