Kastle and Loevenhart' first noted the remarkable inhibiting action by vwm20081

VIEWS: 0 PAGES: 17

									    THE       INHiBITING      EFFECT OF SODIUM                     FLUORIDE           ON
                           THE ACTION OF LIPASE.
                                    (Second Paper.)
              BYA. S. LOEVENHART          AND GEORGE      I’EIRCE.
   (From   the Laboratory of Physiological Chemistry of the Johns Hopkins
                                  VniveYsity*)
               (Received for publication,   November 13, 1906.)
    Kastle and Loevenhart’ first noted the remarkable inhibiting




                                                                                            Downloaded from www.jbc.org by guest, on July 28, 2010
 action of hydrofluoric acid and sodium fluoride on the ester-
 splitting property of liver and pancreas extracts.   Hanriot hart
 previously stated that sodium fluoride does not inhibit the action
 of lipase on monobutyrin.       Arthus3 has since confirmed the
 statement of Kastle and Loevenhart that sodium fluoride inhibits
 the action of lipase. He fouhd that the action of blood serum
on monobutyrin is considerably retarded by this salt. In their
experiments Kastle and Loevenhart studied the effect of sodium
fluoride on the hydrolysis of ethyl acetate and ethyl butyrate by
turbid liver and pancreas extracts.     The experiments with ethyl
butyrate and liver extract were of fifteen minutes’ duration; the
fluoride   and hydrofluoric  acid acted in a dilution of I to 5000.
The per cent. hydrolysis was decreased from 8.54 to 1.13 by the
sodium fluoride and from 8.54 to 0.63 by the hydrofluoric acid.
In connection with other work to be presented later, we were
led to study       more   closely   the inhibiting       action   of sodium      fluoride
on the hydrolysis of fats and lower esters with the result that
our observations seemed sufficiently interesting to warrant
separate publication.
  Experimental  Methods.-Unless otherwise stated the enzymie
preparations used in this work were from the liver and pancreas
of the pig.        In a few instances       corresponding         preparations       from
the dog were employed.              We have used: (I) turbid extracts of
 1 Anzer. Chew. Journ.,    xxiv, p. 491,         1900.
 *Arch. de physiol.. x. p. 797, 1898.
 s Jouvn. a% phytiol., iv, p. 5 6. 1202.
                                           33:
              Effect of Sodium Fluoride                 on Lipase
these organs prepared by grinding IO grams of the fresh tissue
with coarse white sand and extracting               with water.        This was
then strained through cloth and the total volume made up to
IOO cc.      Toluene was always added as the preservative.                  This
preparation      we shall refer to as IO per cent. turbid extract.
 (2)    Clear liver extracts prepared as follows:            The turbid liver
extract just described was placed in the thermostat                    at 400 C.
for about eighteen hours, when coagulated masses appeared.                     Tt
was then repeatedly filtered through an ordinary pleated filter,
when a perfectly        clear highly active filtrate is invariably           ob-




                                                                                    Downloaded from www.jbc.org by guest, on July 28, 2010
tained.     The color of this filtrate varies from a straw color to
bright red. We shall refer to this undiluted preparation as clear
 IO   per cent. liver extract, and in experiments              in which it was
diluted the degree of dilution will be indicated by the per cent.
The corresponding        preparation     from the pancreas has only very
 slight activity, the enzyme apparently remaining in the coagulum.
 We have obtained clear extracts of the pancreas by the method
 proposed by Kastle, Johnston, and Elvove i for obtaining clear
extract of the liver.       One gram of fresh pig pancreas was ground
 up with sand and extracted with 75 cc. of water.                  Then 2 C.C.
 of & sodium hydroxide were added and the mixture warmed on
 the water bath to 380 C. when, 3.5 cc. of & butyric acid were
 added.     This causes a coagulum to separate and on filtering a
 very faintly opalescent filtrate is obtained.             While the filtrate
has some activity it is very decidedly less active than the turbid
 extract.     The experiments        were conducted       with ethyl acetate,
 ethyl butyrate,    diacetin, triacetin, and olive oil. The first four
 were employed in such quantities that for complete saponification
 they would require approximately           40 cc. of & sodium hydroxide.
 That is to say they were employed in acid-equivalent                 but not in
 molecular-equivalent       quantities.      This was done because Kastle
 and Loevenharta found the hydrolysis             to be largely independent
 of an excess of the ester, and furthermore           it has been repeatedly
 shown that the acid produced inhibits further hydrolysis.                   For
 these reasons the esters were employed in quantities which would
yield equivalent amounts of acid on complete hydrolysis.                     The
  * Amer. Chem. Joum.,      xxxi.   p.   52x.   x904.
  1 Lot. cif.
               A. S. Loevenhart                and     George         I’eirce            399
quantities      actually used were as follows: ethyl acetate-0.1747
gram, ethyl butyrate - 0.23 gram, diacetin - o. I 7 I 4 gram,
 triacctin-0.1497        gram.    Three-tenths    of a gram of neutral olive
oil was used in each experiment unless otherwise                 stated.     This
amount would require 21.36             C.C. of & potassium hydroxide            to
saponify it. The titrations           in the experiments       with the lower
esters were made with $‘,, aqueous sodium hydroxide.                     In some
experiments        litmus was used as the indicator, in others phcnol-
 phthalein, no difference between the two being noted in the exper-
 iments with clear extracts.        When turbid extracts were employed,




                                                                                               Downloaded from www.jbc.org by guest, on July 28, 2010
litmus was always used. In the experiments                     with olive oil
  20 C.C. of neutralized     85 per cent. alcohol, made up from absolute
 alcohol, were added at the conclusion of the incubation and the
titration     carried out with & alcoholic potassium                hydroxide,
 phenolphthalein        being used as the indicator.         All the experi
ments were carried out in the thermostat             at 380-400 C. Toluene
 was used as the antiseptic.            Blanks of two kinds were carried
through       with each series : (I)        Experiments     with the active
 enzyme without the addition of ester; (2) experiments                  with the
 boiled extracts to which the ester was added.             The initial acidity
 has always been deducted in the numbers given.                   The amount
of acid formed in these blanks was insignificant                except in one
case which will be pointed out later.
     The numbers given in the tables which follow state the amount
of & alkali in cubic centimeters required to neutraliT& the acid
produced by the action of the enzyme on the ester.               All necessary
deductions have been made. The duration of the experiments
varied and will be given in connection with each series.                  As will
be seen in the tables, duplicate experiments were carried through
in many instances.           The averages of identical experiments            ase
given and these were used in the calculation of the inhibition or
acceleration.         In the tables the concentration         of the sodium
fluoride in the total mixture is given.
EFFECT    OF    SODIUM    FLUORIDE        ON     THE     HYDROLYSIS         OF   OLIVE   OIL
    AND   ETHYL      BUTYRATE        BY   IO   PER     CENT.   TURBID       PANCREAS
                                      EXTRACT.

  Se&s I.-Duration    of experiments, zo hours.  Temperature,   3S” C.
                                                              of
  One cc. of a IO per cent. turbid pancreasextract and 4 C.C. water,
 400                  Effect of Sodium Fluoride                  on Lipase
or 3 cc. of water       and r CC. of sodium        fluoride    solution   were    used     in each
experiment.
    The total    volume     in each case therefore          was 5 C.C.
 _.~---^-.--~--.           _.-.---------_---------
  N~~i&$~-            cmppption                                                          ‘er cept.     of
                                                                                         Inhibttion.
 ____---_-.                           --                                                 --
                                                                                                -
         ;                ii.“0
                                                                                                 Y5
        43                 1l50,000
                           1: 5,000            “                                                13.0
        6                  I:500                                                                31.5
        6                  1:250                                                                35.0




                                                                                                            Downloaded from www.jbc.org by guest, on July 28, 2010
- ..--- -.-. I.._--    _.... .- ---   . . .---..-.-.-.--     _...-~-      __.._._ -      -..-

      In Experiment       I of the above          series the pancreas extract
  showed a large production             of acid on standing even when no
  olive oil was added.         This has always been observed.               It is un-
  doubtedly    to be attributed         to the hydrolysis        of the fat of the
  pancreas itself, which is invariably          present in the turbid extract.
  This large production         of acid in the turbid pancreas extract is
  not observed in aqueous titrations              and is therefore due to the
  higher fatty acids.        As shown in Experiments             2 and 3, the acid
 production     was considerably         increased by the addition of olive
 oil. Furthermore,         the acid production            is decreased by the
 presence of sodium fluoride, I : 2 50 (Exp. 6), to a point below that
 observed in Experiment           I in which neither        olive oil nor fluoride
 was added.       It will be observed that the duplicate experiments
 do not sholv a perfect agreement.            This is probably due to the fact
 that in measuring         these turbid extracts,          in spite of thorough
 shaking before each filling of the pipette unequal amounts of the
 suspended mat.erial were obtained.              The agrcemcnt is sufficiently
 close, however, for our purpose.               Although the differences be-
 tween some of the duplicates are nearly as great as the differ-
ence seen with different concentrations                 of sodium fluoride, yet
 the averages show distinctly             that with increasing dilution the
inhibiting action of the fluoride gradually decreases.                   When the
 sodium fluoride acted in a concentration                 of I: 50,ooo     (Exp. 3)
 practically   no inhibition       (4.5 per cent.) was noted.          It was very
important for our purpose to make an accurate comparison of the
inhibitory    effect of sodium fluoride on the hydrolysis               of olive oil
and ethyl butyratc            under conditions          as nearly identical        as
                 A. S. Loevenhart                   and George Peirce                               401

 possible.    It was necessary in making this comparison to use an
 extract which has considerable action on both of these substances.
 As will be pointed out later liver extract has very little action
 on olive oil, whereas pancreas extract is quite active on both.
 Therefore, two series (II and III) of experiments      were carried
 through    simultaneously    which were identical in all respects
 except that olive oil was used in one series, while ethyl butyrate
 was employed in the other.        It was necessary to use the same
 extract for both series because the effect of the fluoride varies
 to a certain extent with different extracts.




                                                                                                          Downloaded from www.jbc.org by guest, on July 28, 2010
    Series Il.-Duration     of experiments, 20 hours. Temperature,     3P C.
    Two C.C. of a turbid IO per cent. pancreas extract and 3 C.C. of water, or
 2 C.C. of water    and I C.C. of sodium fluoride solution, were used in each
 experiment.      The total volume in each case, therefore, was 5 C.C.
                           _----        I
                                                    _-~--


                                              Increase           in Acidity.
No. of Ex.
periment.                  9live Oil,
                            hms.
                                                                c-3           Averagt
                                                            -      . .--     ____           --
                                                                                              -    -
             tx
             115,000,000
                                            3.1
                                            6.15
                                            6.90
                                                                                              -
                                                                                              -    13
             1: 500,000            I‘       6.30                                              -
             1: 50.000             I‘       6.05                                                     :
             1: 5,000              “        5.20                                              3    -
             1:500                 ‘I       3.10                                              50   -
                           _--                              .__.__..__     I _-_._.     -




 We wish to emphasize that we do not attach much significance
 to small results as the method is hardly trustworthy for small
 differences. There is no doubt, however, that the fluoride,
  I: s,ooo,ooo (Exp. 3), accelerated the process. We have fre-
 quently seen such accelerations when very small amounts of the
 fluoride are used. Similar results have been obtained with the
 esters as will be pointed out. It has been observed by various
 investigators that in many instances a substance which in ordi-
 nary concentrations exerts a powerful retarding influence on a
 process, may in great dilution accelerate it. This has been ob-
 served with remarkable frequency and the case under considera-
 tion is no exception to the rule. This fact deserves recognition.
402              Effect of. Sodium Fluoride on Lipase
   Series III.--Conditions identical in all respects to Series II, except
that ethyl butyrate was used instead of olive oil.
--
                                                      Increase     in Acidity,
                  Concegrgtion         of                                        -__--            Per cent.
                                                                                                  Inhibition.
                                                                                                                  of
                                                                   (2)             AVWilSX!.
                                                                                 ---.-

                    0.0                                          S.QS             9.28
       2.           : : ~~~0~~~00
                                                                 txx
                                                                                  9.08
       43           1 i 50,600                                   7:7s             %
                    1: 5,000                                     2.73             2183
       :            1:500                                        1.38             1.38
-.-




                                                                                                                       Downloaded from www.jbc.org by guest, on July 28, 2010
The comparison of Series II with Series III, in the following
summary,shows that the action of turbid pancreas extract on
ethyl butyrate is inhibited to a much greater extent ‘by the
fluoride than is the action on olive oil.




Thus     the     concentration,             I:   ~,ooo,ooo,            was       approximately                  the
vanishing point of the inhibition with the butyrate, while
I: 5,0&o represented the same point with the olive oil. Viewed
in this way therefore, the inhibiting effect of the fluoride on the
hydrolysis of the butyrate is about one thousand times as great
as on the hydrolysis of olive oil. In some experiments with a
specially prepared pancreas powder we have been able to inhibit
the action on ethyl butyrate to the extent of 85 per cent. by
means of sodium fluoride J: 5,000, while the action on olive oil
showed no inhibition whatever under the same conditions.

EFFECT      OF   SODIUM          FLUORIDR        ON    TBE       HYDROLYSIS               OF   ESTERS           BY
                                   CLEAR     LIVER       EXTRACT.

   The action of clear liver extract on the lower esters proved
to be even more sensitive to the fluoride than the action of the
turbid extracts of the pancreas.
                A. S. Loevenhart                               and George                           f’eirce                       403
     Series IV.-Duration           of experiments,           17 hours. Temperature,     40~ C.
     One C.C. of clear IO per cent. extract of pig liver, 0.23 gm. of ethyl
 butyrate, and 4 cc. of water, or 3 C.C. of water and I C.C. of sodium
 fluoride solution, were used in each experiment.
------_---__--.^--                       -__---.._--.               _..-.-..-
                                                 Xncreasc     in Acidity.
   No. of Ex-      Concefitraption  of     _-.-.    -_---___-     __-...__.-  - Per cmt. of
  periment.                                                                                                         Inhibition.
                                                  ____--(1)                   (2)           -
                                                                                                Average                           -

                   0.0                               8.43                  8.47                   8.45
       i           ; : ~~~~~~0                                                                                             3.5
                                                     73’::             f%                         ::z
       4”          1 i so,doo                        1:os              1:07                       1:OS                      x7”




                                                                                                                                            Downloaded from www.jbc.org by guest, on July 28, 2010
-._--..-.c                          -         ~                 --____---____
    Thus the fluoride when acting in a concentration of I : 500,ooo
depresses  the actions over 50 per cent. but at a dilution of I :s,ooo,-
ooo very little inhibition was noted in this series. The effect of
the fluoride I : 5,000,ooo varied indifferent series. In some cases
it caused an inhibition of as high as II per cent., while in one
instance it caused an acceleration of 6 per cent. The action of
the fluoride varies considerably with different esters of the lower
fatty acids. In this connection we have studied the behavior
towards ethyl acetate, diacetin, and triacetin.
   Series V.--Ethyl acelate. Experiments    were performed                                                      in manner             de-
scribed in previous series. Duration,    18 hours at 40°C.
                                             ,_____...-           --~~-    ---.-     ---            ._.. --,-    _--_____.__.
   No. of        Conce;;tara~.ion       of                    Increase       in Acidity,                          Per cent.       of
Experiment.                                    -._---~                                                              Inhibition.
                                             t       (1)             l--G---lAGGi:
                                                                                                                 --

                  0.0                               4.43                   43’::                 4.81
                  1: 50,000.000                     3.94                                         3.95
                  ; : gpb”,“”                       2.23                   2:08                  2.16
                  1 i 60,dOO                        0.15
                                                    0.65                   0.60
                                                                           0.16                  E
                  1: 6,000                          0.10                   0.13                  0:12

  Series VI.--Diuc&in.                  Otherwise,            same as above.

                                                                                                                  Per cent. of




                                                                      --I--
                                                                                                                   Inhibition.
                                                                      --
                                                                            m                Aver&.
                                                                                           --__

                                                                           2.2
                                                                           1.97                 29
                                                                           1.80                 2:oo
                                                                           1.40                 1.31
                                                                                                          -
404               Effect of Sodium Fluoride                          on Lipase
   Series VII.-Triacetin.              Conditions     same.
                                  --_-~




                                                                                           a
                                                                                           37


 The last three series of experiments show that these three esters
 of acetic acid show about the samedegree of inhibition.    It seems




                                                                                                     Downloaded from www.jbc.org by guest, on July 28, 2010
 to make little difference whether the combined alcohol is ethyl
alcohol or glycerin.      On comparing them with ethyl butyrate
 (Series TV) it will be seen that the hydrolysis of the latter is
inhibited to a much less extent than the esters of acetic acid.
The inhibition with acetic esters is about ten times as great as
with the butyrate ester calculated from the values found with
sodium fluoride, I : 5,000,ooo.
    In order to determine whether the inhibition is influenced by
the proportion of fluoride to enzyme, a series was carried out in
which the amount of enzyme vgried, while the concentration of
the fluoride in all the tubes was the same, viz., I: IOO,OOO.
  Series VIII.---Duration   of experiments, 24 hours. Temperature,     40~ C.
Ten per cent. clear extract of pig liver, ethyl butyrate, Na F in every
experiment,    I :IOO,OOO. The total volume was 5 C.C. in each experiment.
                             --            --.
 No. of Ex-
              I
                    Liver     Na F.
                                                 C.C. Gb NaOH
                                                     0-              Required.        Pet cent. of
 perimant.        Extract.                                                            Retardation.
                                                 (1)      (2)              Average.
              I
                             _-.----_                  -----......     I
                                           11.97         12.32             12.14
                                   -I-      5.87          5.77              5.82          52
                                           10.41         10.56             10.48
                                  -I;       3.26          3.06              3.16          ii
                                            8.48          8.43              8.45
                                             1.86                                         5;
                                  ‘$
                                            5.97           El
                                                          1.86              1.86
                                                                            5.92


                                  5
                                   -I-
                                            :-Et
                                            oh4
                                                                            E
                                                                            0:lS
                                                                                          ii.2

                                                                                          ii.8


From this series of experiments we seethat for a given strength
of sodium fluoride the smaller the amount of enzyme acting the
greater is the percentage inhibition.  This proves that there
                      A. S. Loevenhart                     and George                        Peirce                          405

 exists a quantitative      relationship between the enzyme and the
 fluoride.    ‘V\Tewill return to the significance of this result later.
     By using very dilute liver extract it is possible to show that,
 the fluoride, I : ~oo,ooo,ooo, exercises a distinct inhibiting action
 on the process, as shown in the following.
     Series IX.---Duration     of experiments, 20 hours. Temperature, 39’ C.
 One       C.C. of IO per cent. clear liver extract was diluted        to 16 cc. with
 water and I C.C. of this was used in each experiment         together         with ethyl
 butyrate, and 4 C.C. of water, or 3 C.C. of water and 1 CC of sodium
 fluoride.
                                                                ___.-.-.--~~-.~-_




                                                                                                                                   Downloaded from www.jbc.org by guest, on July 28, 2010
                                                       C.C. of ps NaOH               Required.                       Per cent.
 No. of Ex-       Concerption         of    -_~-~-~               ---._---._                   ------
 periment.                      -.                                                                                  of Ketard-
                                                                                                  Average.              ation.
                -.~--~~-.--          -...   ~- (1)       ..-- (2)            __-___ (3)         --.-.--            -~---_

                0.0                           5.20          5.13       5.05                       5.13
        i         1: 1,000,000,000            5.08          5.10       5.11                       5.10                 OS,
        3         1: 100.000,000              4.95         4.66        5.02                       4.88                  4.9
- -..---_     _ .-.-~~-_-      -.-~ ~.-~~~--.--        ! _..__________
                                                       -                                       _._ ~.~__           ._~~ . .._.__

    Another series of experiments                           was performed                       with         even more
 dilute liver extract.
    Series X.-Duration       of experiments,         I 7 hours.     Temperature,      4r0 C.
One cc. of IO per cent. clear liver          extract     was diluted      to rco c.c. with
water     and I C.C. of this was used in each experiment             together    with ethyl
butyrate     and 4 C.C. of water,      or 3 c.c. of water         and I cc. of sodium
fluoride.

                                                      C.C. of ZS NaOH            Required.
 No. of Ex-                                                                                                  Per cent.       of
 periment.                                                                                                   Acceleration.




 Thus with this very dilute extract the fluoride, I: ~,ooo,ooo,ooo,
 caused a remarkable     acceleration    in the process.      We have
already pointed out that the fluoride when acting in very dilute
solution also accelerates the action on olive oil, but the amount
required to show the acceleration with olive oil would be sufhcient
to very greatly inhibit the action on the lower esters.
   It was frequently noted after titrating a series that those tubes
which contained the fluoride developed acid much faster than the
             Effect       of Sodium      Fluoride       on Lipase

corresponding  tubes in which no fluoride was present.        Thus
TUJXS 3 and 4 of Series VIII were allowed after titration to stand
one hour at room temperature    when they were again titrated.
       No. 3 (no fluoride) required 0.55 C.C. of & NaOfI
       No. 4 (fluoride,    l:lOO,OOO) required   3.10 C.C. of $s NaOlI.
This fact proves that the fluoride does not destroy the enzyme
but merely inhibits its action. * It has been shown repeatedly that
the enzyme is destroyed by acids, and since the tubes containing
no fluoride developed such a large amount of acid during the




                                                                              Downloaded from www.jbc.org by guest, on July 28, 2010
incubation, the enzyme in these tubes was largely destroyed,
The fluoride by inhibiting the action of the enzyme decreased
the amount of acid to which the enzyme was exposed during the
incubation and thus indirectly conserved it. This is probably
the principal reason why the fluoride tubes show a greater acid
production than the water tubes after the titration.     We found,
however, that there is at least one other important factor which
must be taken into consideration here. It is this: The in-
hibiting action of the ‘fluoride increases as acid develops.
This was shown by the following experiments:
             (1) 1 c.c. of clear 10 per cent. extract    of pig’s Iiver.
                4 C.C. of water.
                Ethyl butyrate.
                Litmus.

            (2) Exactly     the same as No. 1.
            (3) ! cc. of the same liver extract.
                3 cc. of water.
                1 C.C. of sodium fluoride (1 :lO,OOO).
                0.26 C.C. of ethyl butyrate.
                Litmus.

            (4) Exactly      the same as No. 3.

   Tubes I and 3 were kept constantly very near the neutral point by
 adding & NaOH as rapidly as they became acid. Tubes L and 4 were
                                       ---
    * Note added during proofreading.-     We have found ‘that the flu&ride
may be completely      removed from the extract by dialysis leaving the
enzyme uninjured.      The activity after dialysis was as great as that of
the extract which hsd not been treated with the fiuoride.
            A. S. Loevenhart        and George Peirce                          407
allowed to stand at room temperature. All of the experiments lasted
36 minutes. The results were as follows:
                                                                    Per cent.     of
                                              Remarks.              Inhibition.
(1) required 5.02 c.c. & NaOH.    Kzpt CL!ne+ral p+nt.                     -
             2;; ::        ‘(
                           “                                             47.8
                                  Ac,‘d alloted    t2 accumulate.
            0.35 (‘        *‘                             “              79.4

   Thus the tubes which were kept as nearly neutral as possible
showed much less percentage inhibition           than those in which




                                                                                       Downloaded from www.jbc.org by guest, on July 28, 2010
the acid was allowed to accumulate.             Hence the inhibiting
action of the fluoride becomes greater as acid develops. The
probable explanation of this is that hydrofluoric acid is a more
powerful inhibitor than sodium fluoride and as the solution be-
comes more acid more hydrofluoric acid is produced.             The fact
that an inhibition     of 47.6 per cent. was noted even when the
mixture was kept neutral proves, however, that sodium fluoride
is a powerful inhibitor in itself entirely independent of the pro-
duction of hydrofluoric acid. Hydrofluoric          acid is quite weak
and even the organic acids can decompose sodium fluoride with
the production of hydrofluoric acid. The difference observed in
the inhibiting   effect of the fluoride on different esters may be in
part attributed to variations in the amount of hydrofluoric acid
produced by the acids resulting from the hydrolysis.             We are
sure, however, that t.his does not explain the very great difference
noted between the esters of the lower and higher fatty acids
because even when the mixtures were kept neutral in the above
experiments, the fluoride I: 50,000 caused an inhibition         of 47.6
per cent. whereas sodium fluoride I:~O,OOO           only very slightly
inhibits the action on olive oil.
    Three facts prove conclusively that the inhibition       caused by
 the fluoride is in no way connected with the precipitation            of
calcium, viz: (1) The amount of fluoride required is so small
that at such dilution calcium fluoride is soluble: (2) Ammonium
oxalate does not inhibit the reaction to any extent: (3) The
addition of calcium chloride does not decrease the inhibiting
action of the fluoride.
    In order to be sure that the inhibiting        action possessed by
the iiuoride is not shared by other salts, a series was carried
408          Effect of Sodium Fluoride on Lipase
through    with the other halides                of sodium    and potassium               and
with   several other substances.
   Series XI.-Duration    of experiments, 21 hours.    Temperature,      38O C.
One C.C.of a xo per cent. clear extract of dog liver, ethyl butyrate, and
4 cc. of water, or 3 C.C. of water and I C.C. of the solution (I : aso), were
used in each experiment. The substance therefore acted in a con-
centration of I : 1250.
                                                         --
                                                          Per cent. of          Per cent. of
                      Substance.                          Retardation.         Acceleration.




                                                                                                Downloaded from www.jbc.org by guest, on July 28, 2010
               Water                                                                -
               NaCl
                                                                                    -ii
               iE                                                                  -
                KC1                                                                -
                KBr                                                                -
                                                                                   -
                                                                                   -
                                                                                   -
                                                                                   -
                                                                                   -
                                                                                   -

                                                                                   27
                                                                                   ii
                                                                                   -i

    Thus we have been unable to find any substance which exercises
an effect on the reaction which is comparable to that of the
fluoride. Several interesting facts are brought out in the table,
however. A comparison of the halides of sodium and potassium
shows that in both cases the bromide inhibits the reaction less
than the chloride or iodide. In the case of halides and the
nitrate practically no difference is seen between the sodium
and potassium salts. The chlorides of calcium, barium, man-
gane’se; and cadmium               all inhibit    the process.           The       results
obtained with ammonium sulphocyannte and thiourea are inter-
esting, The former exercises a marked inhibiting action, while
the latter is practically without effect. Kastle and Loevenhartr

                g Amer.     Chem.      /own.,    xxix, p. 397, ‘go3
               A. S. Loevenhart          and George Peirce.                 409

 fdund exactly the same thing in their work on liver catalase.
 Here also the sulphocyanate            exercised a marked         inhibiting
 effect, while its isomer, thiourca,          somewhat      accelerated the
 decomposition.
     Thus the action of sodium fluoride1 is perfectly characteristic          as
 far as we have been able to determine and the question presents
 itself, What is the mode of action of the fluoride3             Does it act
 on the enzyme or on the ester?          It is impossible, in the present
 state of knowledge of enzyme action, to give all the possible ways
 in which an enzymic process may be affected by a substance.




                                                                                     Downloaded from www.jbc.org by guest, on July 28, 2010
 We may say, however, in a general way that there are at least
 five general modes of action by which a foreign substance (& e.
 a substance which is not essential to the process or a product
 of the reaction) may accelerate or retard an enzymic process.
 (I)    It may react with the enzyme.         The inhibiting and destruc-
 tive’action   of acids, in general, on animal lipase is an instance
 of this kind.     (2)    It may react with the substance upon which
the enzyme acts, the zymolyte?              An instance of this kind i$
the retarding action of formatdehyde           on the coagulation of milk
by rennin.      Bliss and Novy3      found that rennin is not affected
by formaldehyde        but that this substance acts on the caseinogen
changing it to a body which is not readily coagulated by rennin.
 (3) It may react with one of the products resulting from the
process and remove it from the field of action. An excellent
example of this is the accelerating effect of calcium salts on the
action of rennin. The calcium salts precipitate t.he paracasein
as rapidly as it is formed. (4) The solubiiity of the enzyme or
the zymolyte may be altered. (5) The possibility exists that the
substance may react with an intermediate compound formed by

     I Hydrofluoric    acid and in all probability     all soluble fluorides would
 act similarly.
     1 It is necessary to have some general term to designate the substance
 upon which any enzyme acts. The Germans have introduced the term
 “substrat”      and this has recently been used by English and Americafi
 writers in the forms substrate or substratum.              There is certainly nd
logical justification for this term.                   the
                                       We propose term zymolyte because
 it is in keeping with general chemical nomenclature and signifies definitely
what is meant.        It corresponds to “electrolyte,”      etc.
    d Jown. of Exper. Med., iv, p. 47, 1899.
410           Effect of Sodium ‘Fluoride                       on Lipasc
 the action of the enzyme on t.he zymolyte in consequence of
 which its stability may be increased or decreased.
    Kastle and Loevenhastl         found that hydrocyanic        acid accel-
 erates the decomposition          of hydrogen       peroxide   by copper
 sulphate, and Loevenhartz          showed that hydrocyanic          acid ac-
 celerates the oxidation of formic acid by hydrogen peroxide in
 the presence of copper sulphate.          The probable explanation          of
 both of these facts is that the hydrocyanic             acid facilitates in
some way the iormation            and decomposition        of intermediate
products formed in the reaction.




                                                                                  Downloaded from www.jbc.org by guest, on July 28, 2010
    In order to be sure that the fluoride does not react in some
way with the ester in consequence of which the stability of the
latter might be increased, a series of experiments was performed
to determine the effect of fluoride on the hydrolysis          of the esters
by acids and alkalies.       It was found that sodium fluoride does
not inhibit the alkaline hydrolysis of ethyl butyrate, diacetin,
and triacetin      to any noteworthy        extent if at all, and in the
hydrolysis    of these esters by hydrochloric       acid it acts merely as
the sodium salt of any weak acid should.                That. is to say, it
retards the hydrolysis only by using up a part of the hydrochloric
acid and replacing it with the much weaker hydrofluoric                  acid.
 Hence we are justified        in stating that the fluoride probably
does not inhibit the action of the enzyme by reacting with the
ester.
    It is obvious that the fluoride does not inhibit the process
by reacting with either of the products or by altering the solu-
bility of the enzyme or ester.           Therefore but two of the five
possibilities  remain.     Either it must react with ‘the enzyme or
with some intermediate        product.     We have pointed out that it
certainly does not destroy the enzyme.           Furthermore,    if it causes
the inhibition by reacting with the enzyme, we would expect it to
retard the hydrolysis     of all the esters to a proportionate         degree.
But we have found large variations             when different esters are
used. These facts certainly          speak against the view that the
fluoride reacts with the enzyme.
    We have found, however, that the effect of the fluoride increases
  1 Amer.Chem.     Journ.,      xxix, p.    397,   1903.
  a Ber. d. deutsch.    them.     Gesellsch.,      xxxix, p.   130,   1906.
             A. S. Loevenhart            and George Yeirce                 411
as the amount of enzyme acting decreases.              While we cannot be
sure, we favor the view that it reacts with an intermediate
product formed by the action of the enzyme on the ester in con-
sequence of which the intermediate             product is rendered more
stable.     This view seems to us to be more in harmony with all
the facts which we have brought out.
    In general chemicat behavior the fluorides differ widely from
the other halides.       Corresponding      to this chemical dissimilarity
the pharmacological        action of sodium fluoride differs entirely
from that of the other halides.         It is highly poisonous to nearly




                                                                                   Downloaded from www.jbc.org by guest, on July 28, 2010
all forms of life, the fatal subcutaneous             or intravenous        dose
being put at 0.15 gram per kilo for dogs.1 Its pharmacological
action is doubtless very complicated.              Some of its effects are
probably due to the removal of calcium, but this alone apparently
 cannot fully explain its action.             The remarkable        inhibiting
action of sodium fluoride on thcenzymic               hydrolysis     of esters
is the more striking when it is remembered that it is ordinarily
considered a protoplasmic        poison like chloroform,       toluene, etc.,
which does not interfere with enzymic processes.2 Pavy 3 sounded
a warning against this erroneous view and showed that sodium
chloride in I per cent. solution inhibits the action of diastase
on starch about 20 per cent.              Its inhibiting     action on the
hydrolysis     of esters, however,      is incomparably        greater     than
on any other enzymic process as far as we are aware, and the
possibility    naturally   suggest itself that its toxic action may be
due in part to the inhibition of this class of processes in the
organism.       It suggests the possibility that some cleavage of
this type is of vital importance       in the economy.

                                  SUMMA'RY.

   I.  Sodium fluoride inhibits in a remarkable way the hydroly-
sis of esters by liver and pancreas extracts.
   2.  The hydrolysis   of ethyl butyrate     by pancreas extract is
from one hundred to one thousand          times as sensitive to the

  1 Tappeiner, Arch. f. exper. Path. H. Pharm., xxv, p. 203, 1889; xxvii,
p. x08, 1890.
  2Arthus and Huber, Arch. de physiol., Se Ser. iv, p. 651, 1892.
  3 Journ. of Physiol., xxii, p. 391, 1898.
 4=2           Effect of Sodium.Fluoride              on Lipase
 inhibiting action of the fluoride as is the hydrolysis             of olive oil,
  by the same preparation           and under the same conditions.
       3. The action of clear liver extract on the lower esters is much
  more sensitivs to the fluoride than is the action of turbid pancreas
  extract.      Sodium fluoride, Y: ~OO,OOO,inhibited the action of z
 per cent. clear liver extract to the extent of 78 per cent., while
 that of 2 per cent. turbid pancreas extract suffered an inhibition
  of only 5 per cent.
       4. The hydrolysis       of ethyl acetate is inhibited to a greater
 extent by the fluoride than that of ethyl butyrate.               The fluoride




                                                                                     Downloaded from www.jbc.org by guest, on July 28, 2010
 inhibited the hydrolysis          of ethyl acetate, diacetin, and triacetin
 to about the same extent.              Hence the acid from which the ester
 is derived seems to be the principal factor in determining                   the
 extent of the fluoride inhibition.              The inhibition    seems to be
 largely independent of the alcohol from which the ester is derived.
      5. Very small amounts of sodium fluoride (varying with dif-
 ferent esters and extracts}           accelerate the hydrolysis      of the fats
 and lower esters by the extracts.               Thus the fluoride, I: I,OOO,-
 ooo,ooo, caused an acceleration of sg per cent. in the action of
 0.02     per cent. liver extract on ethyl butyrate.
      6. The inhibiting action of the fluoride increases as the tubes
 become more acid. This is probably due to the production                       of
 hydrofluoric      acid.
      7. Sodium fluoride does not influence materially,            if at all, the
 hydrolysis      of esters by alkalies and it only affects the hydrolysis
 of esters by acids as the sodium salt of any weak acid should.
 From this we conclude that the fluoride probably does not
inhibit the enzymic hydrolysis               of the esters by reacting with
the latter.
      8. The inhibiting action of the fluoride cannot be attributed
to the precipitation         of calcium.
     g. The other halides of sodium and potassium as well as many
other substances          investigated     do not possess an action com-
parable in extent to that of the fluoride.            Hydrofluoric      acid and
in all probability          all soluble fluorides affect these processes
similarly to sodium fluoride,
      JO. The greater the amount of enzyme acting the less will be
the inhibiting effect of a given concentration            of sodium fluoride.
           A. S. Loevenhart       and George Peirce             413
This proves that there exists a quantitative    relation between the
enzyme and the fluoride.
   II.  Sodium fluoride does not destroy the enzyme.
   12.  Of the two modes of action to be considered, (I) that the
fluoride reacts with the enzyme, (2) that it reacts with an inter-
mediate product formed in the action of the enzyme on the ester
and resulting in an increase in the stability of the intermediate
compounds, we favor the latter view, because the enzyme is not
destroyed by the fluoride and furthermore because the inhibition
varies to such an extent with different esters.




                                                                       Downloaded from www.jbc.org by guest, on July 28, 2010
   13. We suggest that the substance upon which an enzyme
acts be called the “ zymolyte, ” since this term is more in keeping
with general chemical nomenclature       and is preferable in every
way to the term substrate which has recently been introduced.

								
To top