CHEMICAL METHODS FOR THE DETERMINATION OF
CLINICAL VITAMIN C (ASCORBIC ACID)
DEFICIENCY*
GEORGE J. KASTLIN, C. G. KING, CLARA R. SCHLESINGER AND J. WEST
MITCHELL
From the Medical Department of the Pittsburgh Skin and Cancer Foundation and the
Department of Chemistry (Contribution 403), University of Pittsburgh,
Pittsburgh, Pa.
Chemical methods for studying vitamin C changes in blood,
urine, and spinal fluid, and thereby evaluating the clinical state of
nutrition, were developed following the isolation of the vitamin
as a single substance in 19321. In the same year the dye, 2,6dichlorophenolindophenol, prepared earlier by Mansfield Clark
and associates2 for oxidation-reduction studies, was introduced
by Tillmans and his associates3 for the direct titration of vitamin
C. The method was modified by Bessey and King4 and Harris 5
in 1933, and has since been modified in respect to details of
procedure in a number of laboratories. The reduction is not
rigidly specific for the vitamin, but is subject to interference by
other substances that may be present in tissues having a reduction potential lower than that of ascorbic acid. The vitamin,
however, has a much greater reaction velocity than is displayed
by most of the other compounds at low pH ranges, and its relative concentration is generally sufficiently high that the possible
interfering agents do not significantly alter analytical values.
Suitable clinical methods based on titration with the dye have
opened the field of vitamin C study for widespread clinical investigation. The daily requirements of the vitamin in health,
and to some extent during disease, have been estimated by means
* Read before the Nineteenth Annual Convention of the American Society
of Clinical Pathologists, June 8th, 1940, New York.
Received for publication July 20, 1940.
882
DETEKMINATION OF VITAMIN C DEFICIENCY
883
of the chemical methods with far greater certainty than would be
possible by earlier types of study.
To determine the state of vitamin C nutrition, however, chemical data should be considered in association with the clinical
condition of the patient and the combined considerations must
be based on the fundamental factors which influence vitamin C requirement and utilization by the body. Among these factors are:
(a) Decreased intake of ascorbic acid. The food may be basically
deficient in vitamin content or the vitamin may have been
destroyed during storage, drying, or cooking of the food.
(b) Increased physiological requirement. The high content
of vitamin C in foetal tissue and in mother's milk indicates that
during gestation and lactation there is an increased requirement
of 50 to 100 per eent above normal. The claim that human
foetal tissue can synthesize vitamin C has not been confirmed and
there is much evidence that the claim is ill-founded.
(c) Increased pathological requirement. Infections and toxic
conditions may increase the metabolic requirement. Apparently
hemochromogen formation in the blood stream may destroy the
vitamin by oxidation. The activity of hemochromogens would
be more destructive of the vitamin in the blood stream in the
presence of a high oxygen concentration than in tissues where the
oxygen concentration is lower.
(d) Faulty absorption. Low gastric acidity and delayed
assimilation may permit oxidative loss of the vitamin after
ingestion. Decreased intestinal absorption may also subject
the vitamin to destructive bacterial action.
(e) Administration of drugs. Drugs may destroy the vitamin
either through hemochromogen formation or by other means.
This is a field that has been inadequately studied.
(f) Age factor. There is reason to believe that the relative
requirement per unit body weight is greater during infancy and
youth. The growing organism is more sensitive to the tissue
injury from borderline vitamin deficiency.
(g) Idiosyncracy. Peculiar cases are found occasionally that do
not give normal blood or excretion values under known conditions
of diet or vitamin therapy.
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KASTLIN, KING, SCHLESINGER AND MITCHELL
We shall describe the chemical method which in our experience
has been useful and practical in studying vitamin C blood plasma
concentration, tissue saturation and urinary excretion in patients.
From such observations, plus consideration of dietary history
and clinical condition of the patient, we believe that it is possible
to evaluate the state of nutrition with specific reference to
vitamin C.
THE DETERMINATION OF VITAMIN C IN BLOOD PLASMA
The micro-technic described by Farmer and Abt6 is in our experience reasonably simple and accurate. The method was devised to obviate obtaining
blood by vein puncture. Sufficient test material was obtained from 0.3 ml. of
capillary blood by skin puncture and placed in a special vial with potassium
oxalate to prevent coagulation. To 0.1 ml. of blood plasma, distilled water
0.1 ml. and metaphosphoric acid solution 0.2 ml. were added to precipitate the
protein. Deproteinized plasma 0.2 ml. was then titrated with a standard dye
solution using a micro burette.
The use of capillary blood for the test, although particularly useful with
infants and for frequent examinations of adults, introduces certain errors. By
skin puncture a variable arterial-venous mixture of blood is obtained with
possible admixture with tissue fluids, and hemolysis is not infrequent.
Blood obtained by vein puncture allows for greater constancy in results. A
larger quantity of blood is obtained, which decreases the difficulties and attendant errors in handling minute quantities. Sufficient plasma is also available for checking results. For routine practice we use venous blood and have
increased the individual quantities in the test technique, retaining the principles of the method.
Technic using Venous Blood:
Solutions: (a) 2.5 per cent and 5 per cent metaphosphoric acid. The solutions are stable for approximately two weeks if stored in the refrigerator (Bessey)7. (b) Standardized solution of 2,6-dichlorophenolindophenol, made up
with one tablet of dye or an equal amount of the weighed powder dissolved in
50 ml. of distilled water. Each ml. of the solution is equivalent to approximately 0.02 mg. of ascorbic acid, but the solution should be standardized
against pure ascorbic acid or other suitable standard before use each day. The
solution is stable for about one week if stored in a dark bottle in the refrigerator.
The presence of a small amount of phosphate buffer pH 6.8, increases the
stability of the dye solution. A standard thiosulfate solution provides a good
means of standardizing the dye8.
Apparatus: Micro burette graduated to 0.001 ml. (Supplied by E. H. Sargent
and Co., Chicago, Illinois); white porcelain titration tile; Pipettes (0.2 and 1.0
ml.) graduated in 0.01 ml. divisions; small glass stirring rod.
DETERMINATION OF VITAMIN C DEFICIENCY
885
Collection of blood samples: Obtain 2 ml. of venous blood in a dry syringe,
avoiding hemolysis by preventing unnecessary agitation, suction or tissue
contact. Place in a tube containing 0.2 ml. of potassium oxalate, evaporated
to dryness, to prevent coagulation. Centrifuge immediately. The serum can
also be used from carefully protected clotted blood.
Protein precipitation: Pipette 0.2 ml. of blood plasma into a 15 ml. centrifuge
tube. With the same pipette add 0.2 ml. of distilled water. Add 0.4 ml. of
5 per cent metaphosphoric acid solution. Mix well to precipitate the protein
and centrifuge. Plasma ascorbic acid will usually be retained in this state for
several hours in the refrigerator.
Titration: Pipette 0.2 ml. of deproteinized plasma into a porcelain tile
depression. Fill the mercury controlled burette with standard dye solution.
Dip the curved end of the burette into the fluid in the tile and while stirring
with a fine glass rod, titrate until a pale pink color is obtained which lasts for
5 seconds. The actual titration should be completed in about ten seconds.
Titrate a blank of 0.1 ml. of 2.5 per cent metaphosphoric acid to the same
end point.
Calculation: The number of ml. of dye used for the unknown, minus the
number of ml. of dye used for the blank, times the mg. of ascorbic acid per ml.
of dye, times 2000, equals the mg. of ascorbic acid per 100 ml. of the blood
plasma.
THE DETERMINATION OF VITAMIN C IN THE URINE—MACRO METHOD
Collection of sample: Single specimens are titrated immediately. Twentyfour hour collections are preserved by adding enough 12 per cent metaphosphoric acid to the receptacle to maintain a final concentration of 2 to 3 per cent.
A less expensive but less certain method, is to add 10 ml. of glacial acetic acid
for each 100 ml. of urine, kept in the refrigerator. 8-Hydroxyquinoline is also
an excellent protective agent. Holmes9 has added carbonate to acidified
samples with good results.
Titration: In titrating urines that are not excessively colored it is preferable
to titrate directly into aliquot samples large enough (generally 10 to 100 ml.)
to consume enough of the dye to give good readings. It is less convenient, but
sometimes preferable, to titrate the urine from a burette into 5 ml. of standardized dye solution and 5 ml. of distilled water in an evaporating dish. When
fresh samples are being titrated, acid should be added to give a pH of about
3.0 to 3.5, to minimize interference from reducing substances other than ascorbic
acid.
ADVANTAGES OF THE TESTS
Metaphosphoric acid, first suggested by Fujita and Iwatake10
and adapted to general use in the indophenol dye titration by
Musulin and King11 and Bessey7, protects vitamin C in solution
886
KASTLIN, KING, SCHLESINGER AND MITCHELL
against atmospheric oxidation more effectively than other acids
such as acetic or sulfuric. Mercuric acetate, which has been
used by a number of investigators as a precipitating agent for
the removal of sulfur compounds, followed by H2S reduction,
offers no advantage in our experience. The modification is very
time-consuming, introduces a number of added hazards, such
as removal of excess H2S without reoxidation, and involves an
additional source of error through the formation of non-ascorbic
acid reducing substances. The vitamin is fairly stable in blood
plasma, serum (if there has been no hemolysis) and in most urines,
if kept cold. The technic is applicable to the routine examination of venous blood samples, and with slight variations to capillary blood if care is taken in collecting and handling the samples.
DISADVANTAGES OF T H E TESTS
The titration must be carried through quickly. The end point,
which is not sharply specific for ascorbic acid, is not stable but
fades at an indefinite rate after the ascorbic acid has reacted.
Hemolysis in blood specimens destroys ascorbic acid rapidly by
oxidation; hence careful collection and handling of the blood is
necessary. The test solutions are stable for short periods only,
so that frequent standardization is necessary. In the urine test
other reducing agents, such as thiosulfate and sulfhydryls,
constitute a potential source of error that may become appreciable
when the excretion levels are low. The test requires scrupulous
attention to details in taking samples and in performing the test.
CRITIQUE OF OTHER METHODS
If a photoelectric colorimeter is available its use is desirable
as shown by Bessey7, and Evelyn, Malloy and Rosen12. It makes
possible less interference from other reducing substances and
from nominal amounts of colored or suspended material. Other
dyes such as methylene blue, have been recommended from time
to time but none has proved so satisfactory in general use as
2,6-dichlorophenolindophenol. Spinal fluid has been recommended for analysis but it apparently does not offer sufficient
advantage to offset the greater difficulty in obtaining samples.
DETERMINATION OF VITAMIN C DEFICIENCY
887
Subcutaneous injection of the indophenol dye, followed by observing the time required for decolorization of the local area
has not proved to be satisfactory. Capillary strength tests
based upon either negative or positive pressure for the production
of petechiae, commonly reveal cases of marked vitamin C deficiency, but such tests are neither as specific nor as quantitative
as the chemical tests. Hence, when used alone the capillary
tests are not suitable for the study of marginal deficiencies.
NORMAL VALUES
The normal daily requirement of vitamin C for the prevention
of scurvy has been estimated to be from 10 to 25 mg.13. It is
evident from many investigations, including our own, that 100
mg. is a reasonable minimal standard for health in normal children and adults, and that this amount will normally maintain an
approximate, though not full, saturation of the body tissues.
From analysis of mother's milk it is clear that an infant normally
received 50 to 100 mg. per day14' "• 16. It is reasonable to conclude, therefore, that formula-fed infants should receive such an
intake and that mothers should be provided with approximately
200 mg. per day during the lactation period.
The degree of tissue saturation will be reflected within certain
limits, in the blood concentration of the vitamin if the tissue
balance is stable. The normal blood levels, expressed in mg.
per cent, reported by different workers have varied widely. This
is apparently due in large part to variations in technic and in a
basic concept of what constitutes the "normal" state of vitamin C
nutrition13' 17' 18> 19' 20' 21. Most investigators believe that a
desirable level is 1.0 to 2.0 mg. per cent, and that values above
0.7 mg. per cent are well above the scorbutic level.
Estimations of the normal daily urinary excretion are also
subject to great variation. The common range may be stated
to be 15 to 50 mg., although no definite standard is available12' 22' 23' 24' 25. Urinary excretion is not seriously influenced
by diuresis (Johnson and Zilva)26.
A reasonably definite relationship exists between the blood
level and urinary excretion, especially if the vitamin intake has
888
KASTLIN, KING, SCHLESINGER AND MITCHELL
been sufficiently constant to allow a steady balance to develop.
A depletion of the vitamin C body stores, with resultant decrease
in the blood concentration to a range of 0.7 to 0.4 mg. per cent,
may be considered to represent a state of mild deficiency. Blood
levels below 0.4 mg. per cent, provide preliminary evidence of
severe deficiency. There is an accompanying but irregular
decrease in the urinary output of ascorbic acid with the fall
in blood values.
The statement is frequently made that blood values below
0.4 mg. per cent constitute the zone in which scurvy develops.
Blood vitamin C concentrations in this range, particularly in
adults, are not necessarily associated with clinical evidence of
scurvy, however. A relatively short time is required to develop
a deficiency that is evident by chemical tests, compared to the
time required to develop tissue changes that are clinically recognizable. Vitamin C is not stored in the body tissues to a
degree comparable to the storage of fat soluble vitamins. A
relatively constant daily replenishment is necessary to maintain
saturation, and a transient variation in vitamin intake may be
reflected in the blood level. The latter does not necessarily
reveal information in a strictly quantitative sense regarding
saturation of the body tissues, but is probably the best single
index available.
Single determinations of blood level or urinary excretion are of
relatively little value, with the possible exception of very high
or very low levels. A series of observations correlated with a
record of the dietary intake of ascorbic acid can be of distinct
value.
SATURATION TESTS
A number of methods for determining the degree of vitamin C
saturation have been reported, in which a test dose of ascorbic
acid was given by mouth or parentally, followed by measurement
of blood level response or of the excretion of ascorbic acid in the
urine. Recently we reported27 a method based on a test dose of
500 mg. of ascorbic acid intravenously, with observations on
blood level and urinary excretion over a four hour period. In-
DETEBMINATION OF VITAMIN C DEFICIENCY
889
travenous administration insures maximum uniformity of
utilization of the test dose. This test dose is large enough to
produce unmistakable changes in the blood level of most patients,
and induces sufficient acceleration of urinary excretion to be
measured readily. Our observations and the reports from other
laboratories indicate that such alterations in the blood concentration return to a stable level in about four hours, and that
urinary excretion in this length of time is proportionate to the
urinary excretion in the 24 hour period. The 4 hour test period
is adaptable to ambulatory patients and eliminates the difficulty
of longer periods of urine collection, particularly in children and
in difficult patients.
METHOD
(a) Collect from the fasting (12 to 15 hours) patient a blood sample and
specimen of urine to determine the content of ascorbic acid, (b) Give 500 mg.
of ascorbic acid in 5 ml. of sterile distilled water intravenously in the arm. (c)
Collect the first blood specimen 5 minutes after injection from the opposite arm.
(d) Collect subsequent blood specimens and total urines at 1, 2, 3, and 4 hours.
(e) Plot the curve of blood ascorbic acid (mg. per cent) and ascorbic acid
excreted (mg.) as the ordinate, and time as the abscissa, (f) Compute the
total urinary excretion in mg. and the percentage of the test dose excreted.
The typical normal saturation curve has the following characteristics : (a) The fasting blood level is 0.7 mg. per cent of ascorbic
acid or higher, (b) The five minute blood level is relatively high,
ranging from 4.5 to 9 mg. per cent, (c) The rate of return of the
blood concentration from the five minute peak to the four hour
level is gradual, indicating no great avidity of the tissues for
vitamin C. (d) The four hour blood level is distinctly above
the fasting blood level, (e) Urinary excretion of the test dose is
greatest in the first hour, and the total urinary excretion in four
hours is 40 per cent or more of the test dose.
The typical severe deficiency curve shows marked deviation from
the normal: (a) The fasting blood level is below 0.4 mg. per
cent of ascorbic acid, (b) There is only a slight variable rise of
the blood level at five minutes, (c) The blood concentration
falls rapidly to near the fasting level, (d) The total urinary
excretion ranges from a few mg. to 20 per cent of the test dose.
890
KASTLIN, KING, SCHLESINGER AND MITCHELL
The saturation curves of the normal and severe-depletion
individuals are definite in type. The response indicated by both
blood concentration and urinary excretion is usually associated
with a corresponding dietary intake, though other factors should
always be considered in an interpretation of the findings.
Many tests which indicate a moderate depletion of ascorbic
acid do not conform as nearly as one might expect to the severe
depletion curves. The relation of the fasting blood level to the
four hour blood level is fairly constant, but there are enough
exceptions to the usual response to make this single measurement
an unreliable guide to either saturation or depletion. The exceptions are greatest in the borderline or moderate deficiency
cases.
In our experience the information given by the blood concentration curve and the urinary excretion curve justifies the added
work of testing both series of samples. The hourly excretion
offers a means of detecting the occasional case of low or high
kidney clearance and other types of irregularity. The rate of
urinary excretion may be delayed occasionally even when the
percentage excretion appears to approach normal.
The difficulties of evaluating the saturation tests in patients
in the borderline state of nutrition was shown by tests made during treatment of vitamin C deficiency. The first responses indicating an improved tissue storage of ascorbic acid are: (a)
An increase in the five minute peak level, (b) a decrease in the
rate of fall in blood concentration, and (c) an increase in the total
urine excretion of ascorbic acid. These changes may be observed
before there is an appreciable rise in the fasting blood level (i.e.
above the fasting blood level of a previous test). During depletion there is an early change in urinary excretion, and in the blood
concentration curve, before there is clear evidence of an alteration
in the fasting blood level.
In adults particularly, the saturation tests, as well as the fasting levels cited previously, may show evidence of severe ascorbic
acid depletion when there is no clinical evidence of scurvy.
This observation gives emphasis to the importance of both
chemical and clinical considerations in relation to vitamin deficiency.
DETERMINATION OF VITAMIN C DEFICIENCY
891
In common with other required nutrients that have been
identified chemically, vitamin C is now recognized as having a
much wider scope of influence on nutrition and health than was
attributed to it before the chemical tests came into use. An
accurate history of the dietary intake of vitamins is difficult to
obtain from patients, hence there is a distinct need for the use of
direct chemical tests to supplement the other evidence available.
The extreme instability of vitamin C in most foodstuffs may
readily lead to deficiencies on dietary regimes that on paper appear to be adequate. In borderline cases, the nutritional state
of patients is subject to rapid changes without recognition of a
change in diet, particularly among those in the lower economic
levels and among those who have been subjected to illness. Poor
nutrition, in a non-caloric sense, is more common than is generally recognized.
The functions of the vitamin in a chemical sense have not been
established, nor is there yet a clear picture of the effects of chronic
nutritional deficiency. It is becoming increasingly evident, however, that borderline deficiencies give rise to impaired health.
Experiments with guinea pigs, for example, show clearly that in
the borderline deficiency state, with no external evidence of
malnutrition, sublethal quantities of diphtheria toxin produce
marked injury to the growing tooth structure,28'29 though identical
treatment of adequately nourished control animals does not
induce such injury.
SUMMARY
Methods for the chemical determination of vitamin C in the
blood plasma and urine are described.
The limitations of single fasting blood determination as a
measure of vitamin C nutrition are given.
A clinical method of evaluating the state of nutrition with respect
to vitamin C is described based upon (a) determining the fasting
blood plasma level and urinary excretion, followed by (b) intravenous injection of 500 mg. of asorbic acid, and (c) subsequent
determination of the blood plasma value at 5 minutes and both
blood plasma and urinary excretion values after 1, 2, 3 and 4
hours.
892
KASTLIN, KING, SCHLESINGER AND MITCHELL
The two types of curves (a) vitamin C saturation and (b)
vitamin C depletion are complementary.
Interpretation of the curves should be based on all factors of
the test and should be considered jointly with the dietary history
and clinical condition of the patient, particularly in borderline
nutritional states.
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