the catalytic reactions of blood. the benzidine-hydrogen

THE CATALYTIC REACTIONS OF BLOOD.

I. A STUDY OF SOME OF THE FACTORS INVOLVED IN THE BENZIDINE TEST FOR OCCULT BLOOD.

BY W. G. LYLE, L. J. CURTMAN AND J. T. W. MARSHALL.

(From the Harriman Research Laboratory, Roosevelt Hospital, New York.)

(Received for publication, July 17, 1914.)

The benzidine-hydrogen dioxide test for minute quantities of blood, introduced by Adler, is described in nearly every textbook on physiological chemistry. Despite its general use, nothing very definite appears to be known concerning the conditions under which this test may be relied upon to give consistent results. The lack of uniformity in the procedures which are used in clini- cal practice indicated the need for an investigation of the various factors involved in this test, with a view to establishing the best conditions for carrying out the reaction. Some preliminary work with acetic acid solutions of benzidine’ showed that the reaction was influenced by the following factors: the concentrations of (a) benzidine; (b) acetic acid; (c) hydrogen dioxide; (d) the quality of the reagents; also (e) the order of addition of the reagents; (f) the age of the benzidine solution.

We have found also that, in order to get uniform results, the most scrupulous care must be exercised in cleaning all the glass- ware (burettes, flasks, test-tubes, etc.) employed in the preparation and use of the reagents. Treatment with chromic acid, followed by thorough scrubbing with soap solution and rinsing with redistilled water, answered this purpose.

1 Although alcoholic solutions are recommended in textbooks, preliminary experiments showed them to be unsuitable, for the reason that satisfactory blanks could not be obtained, even when the reagent was prepared with alcohol redistilled from glass. As we found the simpler aldehydes to give strong tests, this failure to obtain negative controls may be due to small quantities of aldehyde, either present in the alcohol or produced by the action of the hydrogen dioside. Alcoholic solutions were, therefore eliminated in this investigation.

445

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446 Catalytic Reactions of Blood. I

An extensive series of preliminary experiments showed that the ordinary technique, when applied to blood at high dilution, was capable of yielding results varying from a decidedly positive test to a negative one, the results depending upon the relations existing between the concentrations of benzidine, acetic acid, and hydrogen dioxide. It thus became evident that the most satisfactory reagent was to be found only by testing a large number of combinations in which the concentrations of the above mentioned substances were systematically varied. In this connec- tion it may be well to mention that with very low concentrations of hydrogen dioxide and acetic acid, positive tests were obtained with tap water, laboratory distilled water, and even with the latter redistilled from glass.2 Throughout this investigation it was necessary, therefore, to control every test by one or more blanks.

The method recommended in most textbooks for making the benzidine test consists in adding to 1 cc. of a saturated acetic acid solution of benzidine, 1 cc. of t,he suspected liquid, and 1 cc. of 3 per cent hydrogen dioxide. This procedure is open to two objections: first, the solubility of benzidine in acetic acid varies greatly with the temperature; second, such a solution, freshly prepared at room temperature, when diluted 1: 3 with water gives a heavy crystalline precipitate which seriously interferes with the test. In our experimental work with solutions of various concentrations of benzidine, it was imperative, therefore, to rule out those which crystallized under the conditions of the test.

Since crystallization may be prevented by an excess of acetic acid, the following experiments were made to determine the minimum amount of acid required for this purpose. A rough deter- mination showed that at 18” the solubility of benzidine in glacial acetic acid is 16 grams in 100 cc. Accordingly, a 15 per cent solution was prepared by dissolving 3.75 grams of Kahlbaum’s benzidine in about 23 cc. of glacial acetic acid, with slight heating,3 and subsequently making up the volume to 25 cc. with acetic

2 In the preparation of the redistilled water used throughout this investigation, a still of which all the parts were glass was employed.

* It was found that after greater heating, benzidine solutions require less acetic acid to prevent crystallization. Prolonged heating, however, is objectionable for reasons given later.


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W.G. Lyle, L. J. Curtman and J. T. W. Marshall 447

acid. If to 1 cc. of this solution, 1 cc. of water be added, crystallization sets in; if 1.5 cc. of water be added, 0.1 cc. of acetic acid must be added to prevent crystallization; if 2.5 cc. of water be added, 0.25 cc. of acetic acid is required. The series of experiments made to determine the quantity of acetic acid necessary to hold various concentrations of benzidine in solution at W’ gave the following results:

Benzidine Acetic acid required to prevent

crystallization at 18” per cent per cent

8 504 6 40 3 25

In our investigation we decided to experiment with reagents containing benzidine in concentrations of 6, 4, 2, and 1 per cent. The amount of acetic acid required to prevent crystallization in solutions of these concentrations was then calculated from the data given above.

With each concentration of benzidine, the acetic .acid was varied from the minimum required to prevent crystallization to a con- siderable excess. Again, with each concentration of acetic acid, the strength of the hydrogen dioxide was varied over a wide range. This gave in all about seventy different combinations. The range of benzidine, acetic acid, and hydrogen dioxide in the reagents experimented with may be seen in the following table:

6ERIE3 BENZIDINE ACETIC ACID6 - ---.___ -

per cent per cent

A. . . . . . . . / 6 41-77 B . . . . . . . . . . . . . 4 28-57 C . . . . . . . . . . . . . . . . . ..__............. 2 1644 D.................................’ 1

-- _i - 8-26

pm cent

0.020-o. 96 0.014-I .37 0.067-2.40 0.033-2.20

’ All the percentages in this work were calculated on the final volume; i.e., after the addition of the solution to be tested and the dioxide. As the ordinary procedure calls for about 15 per cent benzidine solution diluted 1:3 in making the test, the final mixture contains about 5 per cent of benzidine and 31 per cent of acetic acid.

5 Here, as elsewhere, the calculations are based on glacial acetic acid (99.5 per cent), which was used throughout the work.


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As an example of the general procedure employed in the preparation and use of the reagents of various concentrations, the following may be cited: In a small Erlenmeyer flask, 2 CC.~ of 15 per cent of benzidine solution7 were mixed with 1.20 cc. of acetic acid and 3.20 cc. of redistilled water, making a total volume of 6.40 cc. Therefore, 2.56 cc. of t’his mixt,ure cont#ained 0.80 cc. of the original 15 per cent benzidine solution, or 0.12 gm. of ben-

zidine. This amount (2.56 cc.) of solution was then introduced into a test-tube by means of a graduated pipette and 0.20 cc. of blood solution added. (In making the controls, 0.20 cc. of redistilled water was used in place of the blood.) Finally 0.24 cc. of 3 per cent hydrogen dioxide was added, thus making a total volume of 3 cc. This gives a reagent cont,aining benzidine, acetic acid, and dioxide in the following proportions: benzidine (0.12 gm. in 3 cc.), 4 per cent; acetic acid (1.22 cc.* in 3 cc.), 40.5 per

cent;Q hydrogen dioxide (0.24 cc. 3 per cent HzOz=0.0072 gm.), 0.24 per cent.

The results obtained with the various reagents are summarized in t.he following tables:

6 All solutions were delivered from burettes, except where otherwise stated.

’ 100 cc. of this glacial acetic acid solution contained 15 gm. of benzidine.

* That this quantity of acetic acid was present may be seen from the following considerations: 15 gm. of benzidine dissolved in 92 cc. of glacial acetic acid yield 100 cc. of 15 per cent solution. Therefore, 2 cc. of this soIution will contain 1.84 cc. of acetic acid. As 1.20 cc. additional acetic acid were added, the total amount present in 6.40 cc. of the mixture is 3.04 cc.; and in 2.56 cc., the quantity of the mixture taken for the test, the amount is 1.22 cc.

9 This is the total amount of acid used in the preparation of the reagent and includes that in combination with the benxidine.


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W.G. Lyle, L. J. Curtman and J. T. W. Marshall 449

TABLE I.

Series A. 6 per cent be&dine. Blood 1500,000 (0.2 cc. of 1:iOO,OOO blood solution) .I0

REAaENT

Acu41’. ........ Aor5. ......... Aa6. ......... A$3. .......... M4. ..........

Ar3. ......... Ar4. .........

A63. .......... A64. ..........

per cent per cent

41 <0.24 + 41 0.24 * 41 0.48 - Good. 41 0.96 - Fades too quickly. 45 0.18 + 45 0.48 - Good. 45 >0.48 - Fades too quickly. 57 <O.lS + 57 0.18 * 57 0.48 - Fades quickly. 57 >0.48 - Fades quickly. 77 <0.18 + 77 0.18 - Fades quickly. 77 0.48 - Fades quickly

i I A63.

; better than

Comments. An examination of Table I shows that as the concentration of acetic acid is increased, less hydrogen dioxide is required to give a satisfactory blank and that with these higher concentrations of acid, the color fades more rapidly. Reagents Acr5, A/34, Ay4, and A64 were found to be the best of their respec- tive groups. On comparing these reagents simultaneouslv. Ac~5 proved the most satisfactory of the entire series.

lo The blood solutions employed throughout this work were prepared as follows: Blood of normal haemoglobin content was taken from the finger with a blood pipette, the capillary portion of which had a determined capa.city of 58 mgm. of water. This quantity of blood was immediately diluted to 58 cc. with redistilled water, giving a 1 :lOOO blood solution. From this, by suitable dilution, solutions of lower concentrations were prepared. Although the ordinary technique calls for 1 cc. of suspected fluid in a total volume of 3 cc., we preferred in our work to employ a smaller volume of correspondingly stronger blood solution; thus the 0.2 cc. of blood solution 1 :lOO,OOO is equivalent to 1 cc. (the amount of suspected solution employed in the ordinary procedure) of blood 1:500,000.

11 In the notation employed throughout this work the capital letters A, B, C, etc., refer to the per cent of benzidine, the Greek letters to the per cent of acetic acid, while the numerals refer to the per cent of H,Oz.


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TABLE II.

Series B. 4 per cent benzicline. Blood 1:6’00,000.

Ba5. ......... Ba53. ........ Bff6. .........

Bfl3. .......... E!p4. ..........

B-y2 .......... Br3. .........

B62. ..........

Comments. The above table shows that Bor6, BP4, By2, and B62 are the best of the B series, although By2 did not give con- sistently good blanks. Another set of experiments in which the above four reagents were compared indicated that Bcu6 was the best of the B series.

TABLE III.

HzOz

per cent pa cent 28 <0.28 28 0.28 28 0.50 28 0.60 28 >0.60 32 0.24 32 0.64 32 >0.64 41 0.24 41 0.64 41 >0.64 57 0.24 57 >0.64

BLhNK

+ + + - - + - - + - - - -

Good. Fades quickly.

Fair. Fades quickly. Good. Fades too quickly. Fades too quickly. Faint. Fades too quickly.

Series C. 2 per cent be&dine. Blood 1:600,000.

per cent

Co13 .......... 16 C&3+. ........ 16 Cru4 ........... 16 Co15 .......... 16 cs23 .......... 20 cp3 ........... 20 c/34 ........... 20

28 cr2+. ........ 28 Cr3. ......... 28

28 C62 ........... 44 CSS). ......... 44 C62i .......... 44 C63 ........... 44

-

HzOz BL4NB _____- per cent

0.26 + 0.40 * 0.54 - 0.80 - 0.40 *? 0.54 - 0.80 -

<0.40 + 0.40 - 0.80 -

>0.80 - 0.26 * 0.40 - 0.54 - 0.80 -

TEST

Good. Good. Good. Good. Good. Good.

Fair. Good but fades in 5 min. Fades too quickly.

Fair; fades in 7 min. Fair; fades in 7 min. Fades very quickly; gone in

5 min.


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W. G. Lyle, L. J. Curtman and J.T. W. Marshall 451

This series afforded so many satisfactory reagents that it was thought advisable to perform another set of experiments in which only the best of the series were compared.

The results are given in Table IV.

TABLE IV.

Series C. d per cent be&dine. Blood 1:500,000.

h

--

P(

/ -

BLANK

* in 4 min. -

*? in4min. -

-

-

-

-

-

-

TEST

Good green in 5 min. Blue > 0133. Good green in 5 min. >p 2; in 6 min.

1

Faint green in 4 min.; fading in 7 min.

{

Fair green in 4 min. >Cy2$ in 6 min.

{

Fair green in 4 min. ; fades in 7 min.

Fair green in 5 min.; fading in 5 min.

Comments. With concentrations of acetic acid of 20 per cent or over, the test does not develop strongly, and, furthermore, with the higher percentages of acid, the color fades very rapidly. The choice therefore lies in the Q! series. Since Car33 gave suspicious blanks, Ccr4 seems the most suitable reagent of the C series.


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TABLE V.

Series D. 1 per cen.t benzidine. Blood 1:500,000.

REAGENT

Da43. ....... Da5. ........ Dcr6. ........

Dfl2. . Dp31*. ....... Dj34. ........ Dp5. ........ Dyl$. ....... Dy2. ........ Dr3. ........ Dy4 .........

Ds23. D63 . D6393 .......

A -- Pf

l

-

- CETI ACID - ?r cm

8

8

8

8

Hz02

fxT cent

<0.40 0.40 0.54 0.80

10 0.13 10 0.27 10 0.54 10 0.80 16 0.07 16 0.13 16 0.40 16 1.20 26 .07-2.2~ 26 0.27 26 0.53 26 1.00

-

--

0

-

BLANK

+ - - -

+ 6 min. * 8 min. * 8 min. - - - - - - - - -

TEST

Good. Good. Good but fades more quickly

than Dor5.

Good. Strong; fading in 6 min. Fades too fast. Very faint; fading in 8 min. Very faint; fading in 8 min. Fair; fading in 6 min. Strong; fading in 5 min.

Very faint; fades quickly. Faint; fades too quickly. Good; fades too quickly.

To determine the best reagent of the D series, further experiments were made in which only the best of the series D14 were compared. The results are summarized as follows:

TABLE VI.

Series D. 1 per cent be&dine. Blood 1:500,000.

per cent Dcu4$. . . . 8 Blue < &i. Do15.. . . . . 8 Strong blue. Dfl3.. . . . . 10 Blue < CL~+.

The above results show that with blood 2:106, Da5 gave the best test. When, however, blood of concentration 2:107 was used,

r2 \+‘ith blood 10-G Dp3 gives a decidedly better test than Dp4. 13 With blood 10eG none of the 6 series gave a satisfactory test. I4 The tests with the y and 6 series faded so rapidly that these reagents

were eliminated in subsequent experiments.


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W. G. Lyle, L. J.Curtman and J. T. W. Marshall 453

it was found that, while Da44 developed a fair blue in 6 minutes, Dcr5 gave only the faintest test. Da44 was therefore considered the best of the D series.

General comments and conclusions. Throughout this investigation it was found that quantities of acetic acid in large excess over those required to prevent crystallization interfered with the delicacy of the test by increasing the speed of fading of the blue color. The most suitable reagents in the entire series were found among the CY concentrations of acetic acid; i.e., those containing the minimum quantities of acid. It was also observed that an excess of hydrogen dioxide diminished the sensitiveness of the reagent by rapidly bleaching the blue color. On the other hand, with a relatively small quantity of dioxide, satisfactory blanks were not obtained, and, in addition, with blhod, the color developed very slowly. Of the A series Aa! proved the most satisfactory reagent. Bor6 was the best of the B series. Cot4 seemed the most desirable of the C series and Dcr4 the best of series D.15

Comparison of the best reagents. With a view to determining the most sensitive reagent, the best of each of the four series was selected and these were compared. The results of a number of experiments, with these reagents with blood solutions of various concentrations, showed that with blood 2:106, reagent Car4 was the most satisfactory. With blood 1:106, Ca4 developed a good blue, but this color faded more quickly than that given by Dor4$. When blood 5:107 was used, Ccy4 gave only a faint green, while Da43 gave a fair blue. Dot4+ seems, therefore, to be a more sensitive reagent than Cot4. Reagents Aar5 and Bcr6, containing respectively 6 and 4 per cent benzidine, while fairly sensitive, do not compare favorably with the C and D reagents for the following reasons:16

15 All the reagents selected gave perfect blanks in the numerous experiments carried out at room temperature, which varied from E-20”. n-hen, however, towards the end of this investigation, the temperature rose to 24”, doubtful controls’were obtained. This difficulty was obviated by the addition of 0.1-0.2 cc. of glacial acetic acid to the amount of reagent required for each test. Under these conditions no appreciable difference in the sensitiveness of the reagents towards blood was observed.

16 In our study of inhibitors, an investigation which is in progress at the present time in this laboratory, we found that with blood solutions of fairly high concentration to which tannic acid had been added, or with urines


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a. While giving strong tests with blood solutions of moderate concentration, they do not respond to highly dilute solutions.

b. The A and B reagents (containing 6 and 4 per cent of benzidine) are of a decided yellow-brown color which renders the detection of a slight blue tint very difficult.

c. When freshly prepared, reagents A and B do not possess the latitude of the more dilute benzidine solutions, a slight lowering of temperature or a relatively small increase in the percentage of water causing crystallization.

d. They are more affected by a decrease in concentration of hydrogen dioxide, such as may be caused by the accidents1 addition of a slight excess of water; thus, if to a volume of 3 cc. of complete reagent 0.2 cc. of water be added, a doubtful and in some cases a positive test is obtained.

e. They are decidedly more costly, as they contain more benzidine.

Preparation and use of the reagent. For routine work we suggest that the reagent be prepared in the following manner: To 4.33 cc. of glacial acetic acid, contained in a small Erlenmeydr flask and warmed to 50”, add 0.5 gram of benzidine.‘? Heat the flask for 8-10 minutesI in water maintained at 50”. To the solution add 19 cc. of water redistilled from glass.

Into a perfectly clean, dry test-tube, introduce 1.4 cc. of the above benzidine solution, add 0.2 cc. of water or glacial acetic acid,lg then 1 cc. of the fluid to be tested,20 and finally 0.4 cc. of 3 per cent hydrogen dioxide freshly prepared from perhydrol. This procedure gives 3 cc. of a solution containing 1 per cent of benzidine, 0.4 per cent of hydrogen dioxide, and 1 cc. of suspected liquid.

containing moderate quantities of blood, the stronger reagents seemed to give better tests, due probably to the increased active mass of benzidine in proportion to that of the inhibitor.

1’ The purest obtainable; we used Kahlbaum’s for reasons given below. I* The benzidine usually dissolves before the expiration of this time,

but the continued heating is advised tooprevent crystallization in cold weather.

I9 In cold weather the acetic acid should be replaced by redistilled water. 20 For a control add 1 cc. of redistilled water instead of the suspected

fluid.


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W. G. Lyle, L. J. Curtman and J. T. W.Marshall 455

Order of addition of the reagents. We have found that if hydrogen dioxide be added to a solution of blood, the catalytic activity of the latter is rapidly destroyed. That this is the case is shown by the fact that if to 0.1 cc. of blood l:lOOO, 0.2-0.3 cc. of 3 per cent hydrogen dioxide be added and the mixture allowed to stand for several minutes, a negative test results on the addition of benzidine, even though a fresh quantity of dioxide be subsequently added. We have evidence also that a mixture of benzidine and dioxide decomposes rapidly and loses its sensitivity, although no blue color develops. Experiments showed, however, that a mixture of blood and b.enzidine does not appear to deteriorate appreciably within an hour. Therefore, in making the test, the HZOz must be added last; i.e., to the mixture of blood and benzidine solution.

Purity of the reagents. Although both Merck’s “Reagent for Blood Test” and Kahlbaum’s benzidine ran low in nitrogen and contained traces of iron,-this trace of iron may account for the difficulty in obtaining perfect blanks under certain conditions,-we chose Kahlbaum’s because it proved to be of more uniform purity and gave a lighter colored solution. Both Merck’s and Kahl- baum’s “Reagent” acetic acid gave good results, but unless redistilled the “C. P.” grades did not give perfect blanks. Merck’s “Perhydrol” diluted to contain 3 per cent hydrogen dioxide was used throughout this investigation. An examination of the lead- ing brands of dioxides showed that those containing sulphates or acetanilid gave unsatisfactory results.

Age of benzidine solutions. It is generally believed that acetic acid solutions of benzidine rapidly deteriorate in sensitivity towards blood, some investigators*l stating that with certain preparations there is a falling off of 50 per cent in twenty-four hours. We did not find such rapid deterioration in our solutions, however. A 15 per cent solution of benzidine in glacial acetic acid does not lose appreciably in sensitivity for three days; after this time the solutions become so dark that any green color resulting from the test may have been masked. However, with some 15 per cent solutions which chanced to stand exposed to the air for periods of a week or more, excellent tests were obtained with blood 1:106,

21 Ruttan and Hardisty: Biochem. Bull., ii, p. 225, 1912-13.


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Catalytic Reactions of Blood. I

the blanks being at the most only faintly suspicious. With the 2 per cent solution in 18 per cent acetic acid, the results given in the table show that not only is there no deterioration, but in fact an improvement with age, thus confirming the pre- viously mentioned results obtained with a number of old solutions. The statement that benzidine solutions rapidly lose their sensitivity on standing must therefore be taken with reserve.

SUMMARY.

1. An investigation was made of the chief factors involved in the benzidine test for occult blood. Our results, with reagents covering a wide range of concentrations of benzidine, acetic acid, and hydrogen dioxide, show that the sensitivity of the test was greatly influenced by the relative concentrations of these substances.

2. Dilute solutions of benzidine are exceedingly sensitive to pure aqueous solutions of blood, and with these reagents, under the most favorable conditions, we have been able to obtain decided tests with 1 cc. of blood solution of a concentration 1 to 5 million. These reagents, however, are easily affected by inhibiting substances. By increasing the concentration of benzidine, the reagent becomes more resistant to inhibitors, but fails to respond to the more dilute aqueous solutions of blood.

3. The acetic acid serves merely to hold the benzidine in solution; an excess diminishes the delicacy of the reagent by increasing the speed of fading.

4. The hydrogen dioxide functions chiefly in two ways: first, it supplies the oxygen necessary for the reaction; second, it bleaches the blue color. Although a moderate excess of dioxide accelerates the reaction, it interferes with the delicacy of the test by increasing the speed of fading. Moreover, an excess of dioxide interferes with the reaction, both by destroying the catalytic power of the blood, and by reacting with the benzidine itself, with the for- mation of product,s which appear to have an inhibitory action. Therefore the concentration of the hydrogen dioxide should be reduced to the minimum compatible with the production of a blue color which will reach its maximum intensity within 5 or 6 minutes.


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W. G. Lyle, L. J. Curtman and J. T. W. Marshall 457

5. To obtain consistent results, water redistilled from glass should be used exclusively, and controls should invariably be made.

6. It has been shown that in making the test it is imperat,ive that the hydrogen dioxide be added last.

7. Certain grades of hydrogen dioxide are unsuitable for the test; the best results were obtained with Merck’s “Perhydrol” reagent grade. It is also essential that the benzidine and acetic acid be of the highest purity obtainable.

8. Dilute acetic acid solutions of benzidine may be kept for several days without deterioration.

We expect to publish shortly the results of an investigation of certain substances which appear to inhibit the catalytic reactions of blood, and also methods which we have devised for overcoming these difficulties in the examination of body fluids for blood.


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MarshallW. G. Lyle, L. J. Curtman and J. T. W.

BLOODBENZIDINE TEST FOR OCCULT

FACTORS INVOLVED IN THEBLOOD: I. A STUDY OF SOME OF THE

THE CATALYTIC REACTIONS OF

1914, 19:445-457.J. Biol. Chem.

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