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THE ESTIMATION OF FORMIC ACID IN THE URINE. During the by linzhengnd

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									   THE ESTIMATION                      OF FORMIC            ACID       IN THE         URINE.
                    BY   ETHEL     M. BENEDICT          AND   G. A. HARROP.
(From        the Department    of Medicine      of the College   of Physicians       and Surgeons
        of   Columbia University,       and the Presbyterian        Hospital,       New York,
               and the Chemical       Division,       Medical    Clinic,      the   Johns
                   Hopkins     University and Hospital,               Baltimore.)

                         (Received for publication,      August 23, 1922.)
   During the course of some clinical and experimental studies of
acute methyl alcohol poisoning (1) the necessity arose for making
a considerable number of quantitative estimations of the excre-




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tion of formic acid in the urine. The method at first employed
was that of Dakin, Janney, and Wakeman (2), which is satisfac-
tory but very time-consuming. It was found desirable to devise
a simpler and quicker procedure, which would obviate if possible
the necessity for extraction with ether and shorten the time
required for the other manipulations. The following method has
proved to be satisfactory in our hands and yields better results
than are obtained by the ether extraction method.

                                            Methods.
   100 cc. of urine are accurately measured into a liter volumetric
flask containing 500 to 600 cc. of water. 100 cc. of 20 per cent
CuS04 are added and the whole is well mixed. A 10 per cent sus-
pension of Ca(OH)2 is then added until alkalinity, as evidenced
by the characteristic color change from green to blue, occurs.
It is well to avoid an excessof calcium hydroxide.      The contents
are t.hen made up to volume and mixed. After standing 15 to 30
minutes they are filtered and a 600 cc. aliquot is placed in a 800 cc.
Kjeldahl flask, with 2 to 3 drops of phenolphthalein as an indi-
cator, and made distinctly acid with 85 per cent phosphoric acid.1
Sufficient excess should be added to free all of the formic acid
present (1 to 2 cc. are ample for this purpose). Glass beads are
   ’ Tartaric acid may be used instead of phosphoric, but we have not found
that it has any particular advantage.  Strong mineral acids cannot be used.
                                                443
444                            Formic             Acid in tho Urine

added to prevent bumping.         This flask is connected to a supply
of steam and to a water condenser by means of a Kjeldahl trap.
Both the neck of the flask and the outside of the trap are well
wrapped with asbestos cord to prevent loss of heat.2 The distil-
late is caught in a casserole provided with 15 to 20 cc. of 0.1 N
NaOH, and a few drops of phenolphthalein              are added to make
sure that it is alkaline.      This alkalinity      must bc maintained
throughout the distillation by the addition of further 0.1 N NaOH
as may be necessary, but a great excess must be avpided.              It is
neither necessary nor advisable to cause the delivery tube from
the condenser to drop under the liquid in the casserole.               The




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distillation   must be so managed t,hat at first a slow stream of
steam is conducted in, while the contents of the distilling flask
 are rapidly driven over by brisk heating and reduced in volume
 to 50 to 75 cc. At this amount they must remain until 2 liters
 are collected.”
    The distillate is t,hen evaporated to dryness over night on t’he
 water bath, and the residue taken up in exactly 100 cc. of distilled
 water and filtered.     A 90 cc. aliquot is placed in a 250 cc. Erlen-
 meyer flask and made just acid with 0.1 N HCI.              10 cc. of t,he
 special HgClz mixture4 are then added and the flask is fitted with
 an air condenser and heated in a boiling water bath for 1 hour.
 After cooling the mercurous       chloride is filt.ercd into a weighed
 Gooch crucible, washed with 100 cc. of 5 per cent cold HCI, then
 mater, alcohol, and ether, and dried 1 hour at 105’ and weighed.
 The blank of the reagents in our experiments has varied from

     2 Such a device is essential,                as otherwise            phosphoric       acid is merhauically
carried      over during           the rapid      distillation          and causes much trouble.                      It is
convenient         to do the steam distillations                     on a Iijeldahl         rack.
     3 This reduction            in volume         is necessary            because      the amount           of formic
acid carried        over by steam distillation                     varies      enormously         with the volume
of the distilling          fluid.        On the other              hand,       if the volume           is allowed         to
become too low, especially                  if it boils dry, large errors result from the break-
ing down of other             organic       substances           present       into formic       acid.      2 liters      of
distillate      are sufficient          to collect          if these precautions               are observed.              It
should      be collected          in about 2 hours.
     4 This solution         contains        200 gm. of HgCl*,                80 gm. of NaCl,            and 300 gm.
of Na acetate          to 1 liter of water              (3).        10 cc. of this solution             are suf%cient
for amounts          of formic        acid up to 0.13 gm., that is a concentration                               sisteen
times      the upper          limit      in normal            urine.        For urines         containing          larger
amounts,        larger    amounts          of this mercuric               chloride    mixture         must be used.
                 E. M. Benedict                   and G. A. Harrop                                445

0.0014 to 0.0044 gm. according       to the reagent used. The
amount of formic acid per liter in the original urine is then 1 01
  lo
x TX;             X 0.0975 X (weight                     of precipitate-       weight      of blank
of    reagents) .5

                                           DISCUSSION.


    As has been frequently     pointed out, the isolation of volatile
fatty acids by steam distillation direct from acidified urines often
entails serious errors, because the prolonged action of mineral




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acid upon carbohydrates       and other substances present, results
in fatty acid, especially formic acid formation.6       The precipita-
tion with copper hydroxide, a device introduced by Van Slyke in
his well known acetone body methods (4), disposes of this difficulty
by removing the sugar and other interfering          substances.      The
steam distillation    should be commenced at once on the filtrate
and its reduction in volume effected in the process.        It is a waste
of time first to try to concentrate the alkaline aliquot and it may
cause considerable error because of the destruction            of organic
acids which takes place when they are heated in alkaline solution.
Further,    it has been found that when formaldehyde           is present
 (as is the case in urines obtained during acute methyl alcohol
poisoning), about 10 per cent will be oxidized to formic acid in
the process of evaporating in alkaline solution.
    The addition of NaCl and of Na(CH&OO)          to the HgCL solu-
tion hastens very greatly the precipitation of HgCl and also pre-
vents the precipitation of impurities.   As seen in the curve (Fig. l),
99 per cent is precipitated after 1 hour heating, at the end of which
the reaction is stopped and 1 per cent added to the final figure of
the result.    We have spent a great deal of time in efforts to develop
a method for titrating the mercujr, but we believe the gravimetric
estimation is more reliable and satisfactory.
    6 1 gm. of HgCl equals 0.0975 gm. of formic                  acid.
    6 It may be pointed           out that this error obtains          in the Ryffel     method     for
estimating      lactic    acid, (i.e., steam distillation         from urine to which           equal
part?     of concentrated        H&30,    have been added),          when determinations            are
made upon         urines     containing     carbohydrates.          Treatment       with    Cu(OH)z
will obviate      this error      in such lactic     acid determinations.
446                        Formic Acid in the Urine
   Determinations   of the formic acid content of urines should be
commenced as soon as possible after collection.    We have found
it quite impossible even when kept on ice or when preservatives
are used, to get concordant results when specimens stand over 24
hours.
   Tables I and II indicate how completely known formic acid can
be recovered from pure aqueous solutions and from urine by this
method.

 Per
cent r
  90




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  80
  70




Min.0 0             15         30         45       60             75         90         105         120
                                                 Time
    FIG. 1. Curve        showing    the   rate   of   formation        of   mercurous    chloride
precipitate.


               The Occurrence of Formic Acid in Human Urine.
   A number of analyses of the formic acid content of normal
urine from individuals on mixed diet are given in the literature.
The amount is small and is given by Dakin, Janney, and Wake-
man, as varying from 29.9 to 118.6 mg. per 24 hours. Our
results are in agreement with these figures.
   A number of analyses on urines from patients which show a
high organic acid content are given in Table III.   It is known
that an increase in the consumption of carbohydrates causes an
increased formic acid excretion, a condition which would also
presumably result from administration of glucose intravenously
                                                         TABLE               1.
                       Recovery        of Formic         Acid      from             Aqueous          Solutions.
                              -                          -
                                        Amount  of                 Amount             of
                                  fcnnie acid added           L wmio acid           found
                                                         .-
                                            l7m.                      om.                              l7m.             per cent

       July       7, 1920               0.2494                     0.2364                            0.0040               1.6
                                        0.2404                     0.2491                            0.0003                   0
                                        0.2404                     0.2442                            0.0038               1.5
       June      10, 1920               0.1202                     0.1198                            0.0004                   0
                                        0.1202                     0.1215                            0.0011               0.9
                                        0.1202                     0.1216                            9.0012               1.0
         “       14, 1920               0.6010                     0.5821                            0.0189               3.0
                                        0.6010                     0.6035                            0.0019               0.3
                                        0.6010                     0.5835                            0.0175               2.9




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         “       15, 1920               0.0060                     0.0064                            0.0004
                                        0.0060                     0.0064                            o.cmlO4
                                        0.0060                     0.0970                            0.9010
         “       21, 1920               0.6010                     0.5904                            0.0106               1.8
                                        0.6010                     0.5948                            0.9062               1.3
       May        9, 1922               0.1208                     0.1218                            0.0010               0.8
                                        0.1208                     0.1203                            0.0005               0.4
                                                         -

                                                      TABLE             II.
                            Recovery         of Formic           Acid             Added       to Urine.

         Date.                          Subject.                        Amount
                                                                        in urine.

                                                                        -I


June      22, 192(                          A

                         Nephritic             with   slbu-                                 0.0240       0.02360.0004           1.9
                           minuria.                                                         0.0240       0.02290.0011           4.8
                                                                                            0.6010       0.60430.0033           0.5
                                                                                            0.6010       0.61270.0117           1.9
                                                                                            0.6010       0.58960.0114           1.9

Oct.     29, 192c                           l3
                         Diabetic,         1.0 per       cent                               0.0240       0.02480.0008           3.0
                            sugar.        Acetone         and                               0.6010       0.59920.0018           0.3
                            Diacetic        ++++

May          5, 1922                       C                            0.0038
                                                                        0.0040
                         Diabetic,        3.0 per        cent                               0.1208      0.11670.0041         3.4
                            sugar.       Acetone          and                               0.1208      0.11700.0038         3.1
                            Diacetic      + + ++

Apr.     19, 1922                          D                            0.0019
                                                                        0.0026
                        Normal         individual.                                          0.1208      0.1167,0.9041
                                                                                 TABLE       III.
                                          Excretion of Formic Acid in the Acidosis of Starvation and oj Diabetes.

                           0.1 N organic     acids.’    0.1 N formic    acid.   0 1 r4 acetone       bodies.      0.1 N acid not
Nnme.                                                                                                            accounted   for:      Blocd                                  3
            Dab?.                                                                                                                      co*.             Condition.
                             Per liter.      Total.    Per liter.      Total.   Per liter.          Total.     Per liter.    Total.                                           B
                          ---P----P                                                                                                                                           c3’
             L!l.%?             EC.            cc.         cc.           cc.        cc.               cc.          cc.         cc.       wz.
                                                                                                                                      per cent
                                                                                                                                                                              5
 E        Apr. 19              488             908        5             9.3         225               418        145          270                Epileptic, starvation.       FL;
 F        June 2           1,576             2,459        9.6          15           932             1,452        433.4        637      23.6            “         I‘
                                                                                                                                                       “         ‘I           F.
 F         “     5         1,706             1,963        4.5           5.2         919             1,057        537.5        620.8                                           s
  s        “     5         1,266             2,472       10.5          21.5         694             1;430        335.5        690.5                    I‘        “
 H        May 29             374.4           2,334       17.6          58           468             1,547        347.6      1,145      27.9      Diabetes, acidosis.          E
 H        June 15            534             2,295       14            60           225               968        251        1,031      33.4            LL        ig
 L         “20             1,552             3,107       16            32.5         935             1,339        446          872      24.0            “         “

   * Creatinine       and creatine have been corrected for in the “acids not accounted for” but not in the total organic                                             acids.




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                 E. M. Benedict                and G. A. Harrop                                449

or by other routes.   The data, however, indicate that an increased
excretion of formic acid does not commonly occur in the acidosis
of diabetes.   An opportunity  to study the excretion in the acidosis
of starvation was afforded by the analysis of urines from epileptic
patients under treatment.     The data indicate that formic acid
excretion plays no part in the production of the high organic acid
excretion usually found in these fasting patients.
                                          TABLE        IV.
   Excretion      of Formic Acid in Experimentat Methyl Alcohol                   Poisoning.
                        Dog K. Weight 7.2 kilos. Male.




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   Date.           Urine.    0.1 NW-                                   Remarks.
                            :anic acid.

    1,920             cc.       cc.            cc.
  May       3       240        271                23
    ‘I      4       600                           24
    “       5       290        250                15         Given 8 gm. CHsOH             per kilo
                                                               by stomach tube.
    “        6      650       468             170
    “        7      275     1,234             532
    “        8      195     1,249             514
    “        9      175     1,929             702
    “       10      200     1,625             516
    “       11      395     1,292             671
    “       12      405       871             266
    “       13      104       807             245
    ‘(      14      380       541             188
    “       15      300       511              51
    “       16      410                        19
    “       17      310        299


  The one pathological condition in which formic acid excretion
has been found to play a predominant role is in the acidosis of
methyl alcohol poisoning. In Table IV is given the formic acid
excretion in one experiment following ingestion of methyl alcohol
by mouth.
                                          SUMMARY.

   An improved and simplified method for the estimation of
formic acid in urine is described.
   No increase in the excret,ion of formic acid has been found during
the acidosis due to diabetes, and in that due to starvation.     The
                   Formic Acid in the Urine
formic acid excretion and its relation to the total organic acid
excretion are presented from an experiment in the production of
acute methyl alcohol poisoning.
                             BIBLIOGRAPHY.

1. Harrop, G. A., and Benedict, E. M., J. Am:Med. Assn., 1920, Ixxiv, 25.
      Palmer, W. W., and Harrop, G. A., PTOC. Am. Sot. for Ciin. Znveslz’ga-
      tion, J. Am. Med. Assn., 1920, Ixxiv, 1669.
2. Dakin, H. D., Janney, N. W., and Wakeman, A. J., J. Biol. Chem., 1913,
      xiv, 341.
3. Fincke, H., Biochem. Z., 1913, li, 253.
4. Van Slyke, D. D., J. Biol. Chem., 1917, xxx, 347.




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