STUDIES ON THE METABOLISM OF
HYDROOUINONE

THESIS FUR THE DEGREE OF M. S.
Robert Pennell
1932

 

     

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ifrhonin summittca.to tho ruoulty of
moms“ Stat. 0011.3. for Partial
mull-ant or the Rocnirunnta of the
Bmsroo of listor at Scionoo

Hobart Roman

Juno, 1982

”2 v-
c 7') f.

The author wishes to express his
indebtedness and gratitude to Dr. C. A.
Hoppert, Associate Prof. of Chemistry,
for his timely advice and assistance in
the pursuit of this problem and in the

preparation of this manuscript.

1 (,3 53 3 :3

INTRODUCTION

Work has been done in this laboratory and
elsewhere (1, 2, unpublished data) showing that hydro-
quinone, when added in small quantities, will prevent
the oxidation of certain readily oxidizable foods. It
was found that certain foods could be stored for long
periods without undergoing oxidative change, if they
were first thoroughly mixed with a small quantity of
hydroquinone. It has also been shown that this chemical
will prevent the destruction of vitamins A. The commercial
use of hydroquinone in this role as a preservative would
supplant at least in some cases the present bothersome and
expensive methods of preservation, such as vacuum pack-
ing. In view of this it was thought advisable to under-
take a study of the fate of hydroquinone in the animal
body.

No previous work on this subject has been re-
ported in the literature. There have, however, been
reports on several closely allied compounds, benzene
(3,4,5,6,7,8,9,), halogen derivatives of benzene (9.10.11),
phenol (10,11,12), benzoic acid (13), acetophenone (6),
and fatty aromatic compounds (14).

The results of these researches may very well
have some bearing on the problem at hand. Thus, it has
been found that when benzene is fed or injected, hydro-

quinone and pyrocatechol may be eliminated in the urine

in amounts large enough to permit isolation and
identification (15). Preusse (16) found that paracresol
when fed to dogs is in part excreted as paracresol
(ester) and is in part oxidized to paraoxybenzoic acid.

Jaffé (8) found that upon administering ben-
zene it was possible to isolate the straight chain
muconic acid from the urine, indicating that the benzene
nucleus had been split. He fed 60 grams of benzene in
lots of 3 grams per day and recovered approximately 3%
of this as muconic acid. He could isolate no muconic
acid when benzene was not fed. Upon injection of 8
grams of muconic acid as the sodium salt subcutaneously
in four doses in the course of 12 hours, only 1% was
recovered in the urine. He believed this to show that
muconic acid itself was readily oxidized by the animal
body. Fuchs and v. 8068 (4) also found that when

3 - 5 grams of benzene was administered daily to leukemia

patients, muconic acid could be isolated from the urine.
Mori (l7) contrary to the results of Jaffé,
found that a large portion of the muconic and adipic
acids administered to dogs was excreted unchanged in the
urine. When injected subcutaneously, from 71.4% to
74.1% of the muconic acid was recovered in the urine.
When given by way of the stomach, 43.6% was recovered.

Neumaerker (3) was also unable to duplicate the work of

Jaffé and Fuchs and v. 8053. He injected benzene in'

doses of not more than 5 grams per week and could
isolate no muconic acid. Upon injection of 4 grams
of muconic said he recovered from 55.1% to 67% of
it unchanged in the urine. He concluded in accordance
with Kori and contrary to Jafle’, that auconic acid was
not easily oxidised in the body.

rhierfelder and Klenh (6), however, correlated
the above results, showing that if sufficient benzene
‘wcre ted or indected and the absorption into the body
were rapid. muconic acid could be isolated in the urine.
They believed the differences in sueonio acid.oxidation
round by Jarre'ae against leuuaerker and Ieri to be due
to concentration.ot the solution injected, differences '
in the weights of the experimental animals as well as
individual differences of the animals thsssslves.

. Underhill and Harris (5) reported that bensene
”acts not only on.the blood.elenente but exerts a catabolic
influence on the body tissues as e.whele, as manifested
by a sharp rise in.oreatinine and total nitrogen within
a very short period atter its subcutaneous injectionfl.

Several investigators have found that sulphur
netsboliss.was disturbed by administration ot‘bensene
or its derivatives. Callow and Hele (9,18) found that
upon feeding aono- and di-ohlorobensene they were
excreted in part as chloro-phenylhercapturie acid and in
3; 01-830

part as o lhey found that this caused an

6 4'

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increase in the 8/! ratio of the urine. rhis they
explained by suggesting that the sulphur metabolism was
hastened and the nitrogen of the catabolised pretein was
excerted later. Toluene and o-chloro toluene showed no
such effects.

Rhoda (ll) administered (.2 g. per kg.)
phenol simultaneously with cystine, taurine and lagsos.
The percentages excreted as ethereal sulfates were 83%.
17% and 27% respectively. Inorganic sulfates and thic-
sulfates were without apparent effect. When bronco-benzene
and di-brono-bensene were fed they appeared in the urine
partly as ethereal sulfates, but when cystine was given
simultaneously they appeared as aereapturic acid.

Shirle, Huldoon and Sherwin (10) found that a
pig reduced to a condition of endogenous l cgtaboli-
and maintained on a carbohydrate diet, excreted about
dag. of ethereal sulfates per day. The animal was then
fed «36353:, causes and p - 0,3403 01. The output of
ethereal sulfates was very decidedly increased in each
case, evidencing the formation of ethereal sulfates from
endogenous sources. The feeding of inorganic sulfates
along with each of the toxic substances resulted in no
increase in the elimination of mlfates. Oystine, how—-
ever. together with each of the same aromatic poisons,

caused an increase in the excretion of this fora of

sulfur with canton, but a decrease with the other two.

 

n.
04

 

lorecver, with 0335 hr. the decrease was accompanied by
a corresponding rise in neutral sulfur. They concluded
that there were two ways of detoxieating phenolic
substances; one, by combining the poison with a sulfate
radical, which is obtained by tissue destruction; the
other by utilising exogenous cystine, forming eventually
a nercspturic acid. this mercepturie acid may be
excreted as such, thereby adding to the neutral sulfur
fraction and lessening that of ethereal sulfates, or it
m be oxidised to a sulfate and increase the output of
ethereal sulfates.

rolin and Denis (19) reported that the dis-
tribution of phenols between the free and conjugated
forms is virtually the sue in animals and man, the free
phenols representing from 80$ to 901 of the total. they
also reported ( :0) that the anount of phenol excreted in
the faces is as hell as tp be negligible. Dubin (21)
in repeating the work of Delia and Denis corroborated
these results. He also found phenols to be increased in
the urine and the ratie of combined phenol to free phenol
to be increased following withdrawal of water, intestinal
obstruction or pancreatic insufficiency. After feeding
l as. of 2h 08 or p-cresol to dogs weighing about 10
kilos. about 66% end 40$ respectively were eliminated as
phenols in the urine.

these reports on bensene and its derivatives,

although not directly applicable to the probl- at
hand, gave a idea as to what one might expect upon
feeding hydroquinone. it the ease time they indicated
the direction in which to proceed with this probln.

W

L pig was selected as the experimental animal
with the idea in mind that the metaboli- of swine
approaches that of buses mere elesely than does the
metabolism of ether animals. fhe animal selected was
a young pig of somewhat less than 100 pounds weight.

A cage was made for the metabolism work, con-
sisting of two parts, the sage proper, and the feeding
cage. The cage proper was 4' x d' x 5' in dimensions.
lhe cage was completely sine lined. One side of it
was hinged to permit easy access fer cleaniu. the top
of the cage was covered with iron bars spaced about 6'
apart. fhe cage was flecred with heavy iron screen.
no cage was on casters and was placed on a platform
about a foot and a half in height. the platform was twice
the length of the cage, one half of the platform consist-
ing of a drain beard covered with metal. Ibis drain

elected at the cue angle from each side, and at the
center there was an opening under which a bottle was
placed for the collection of urine. The cage steed ever

the drain except when being cleaned. It could then be
run from cvcr the drain and the drain could be washed
and scrubbed. the teding cage was li' x d' x 5' in
nice. the sides of this cage were also cinc lined and
it was roofed with iron reds. rho feeding trough,
sinc lined, was placed at one end of the cage. d small
door opened ismediately above the trough to permit
mixing the food. !he floor of the back part of the
cage consisted of heavy metal screen. Under this was s
sine-lined draIer to receive any urine voided while the
animal was in the feeding cage. the two cages were
connected by doors mich could be raised or lowered at
will. the cages were securely fastened to each other
by hooks.

the diet selected for the crperiaental animal
was a balanced ration made up as follows: 701 corn
meal, '10; whole mill: powder, 10% oil heal, a; alfalfa
meal, 15 la 61 and 1’ bone ash. On this diet the daily
output of phenols remained fairly constant. he animal
received 500 us. of the ration per do. It was fed by
mixing well with water in the trough. the hydrocuinons
was given with the food by dissolving it in the water
added to the food mixture.

Animal I was placed on the above diet for two
weeks before hydroquinone was fed. thenols were detersined

daily on five consecutive days of each week during the

experiment. a. question arose as to dust method should
be used in the detenination of phenols. rhe method of
tom and Denis (88.80.19, 19.80.31.81,58,3d,85) was
“1...“ 1. spite of the fact that a nubcr of
investigators have found it to be non-specific (28, 88,
8d. :6, 26, 87). A careful review of the literature failed
to reveal another method which would adopt itself to
daily routine. d very good review of the literature of
phenol detersinations up to the year 1926 is given by
Gibbs in chcmical Reviews (86).

the results of these analyses are given in
table I. It will be seen, taking the figures of the
first two weeks as a basis, that a little more than three-
fifths of the hydrequinonc was excretcd~daily as fed.
this appeared in the urine both as free and conjugated
phenol in about carnal proportions. During the sixth
week when 3 gas. of hydrequinone was fed daily the pro-
portion of conjugated phenols was increased slightly.
rhis night indicate a special power of dctoxication in
the body in the presence of extraordinarily large
quantities of phenolic substances. after discontinuing
the feeding of hydroqsinonc it will be noted that the
phenol content of the urine did not return at once to
normal. i'he phenol excretion for the first three weeks
after the ministration of hydroquinone was stopped was
definitely higher than that of the two weeks control period

preceding the feeding of hydroguinone. here was a
gradual decrease towards the normal val use, however.
Animal I differed fron all the ensuing aninals in this
respect.

these data would soon to show that the greater
portion of the hydrcquinene was excreted as fed.
dpparently, however. none of the material was stored in
the body and was excreted after the feeding of hydro-
cuinone was discontinued. Using the control period as
a basis, approxinstely 75$ of the hydroguinone was excreted
as free and conjugated 'phcnols'. rho animal showed no
apparent detrimental «at. as'the result of this
Wrinent.

 

 

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26

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‘18.. th mr'?!at.1 p5.

670.6
265.26
660.6
581.4

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, 0

191.6
356.6
840.6
866.9
267.8

 

616.4
886.8
666.6
606.6
860.8

 

‘3:I7Uii:*

669.6
630.2
742.8
1,864.1
610.06

 

1. 873.6
401.6
704.4
967.3
608.8

 

 

915.5
1,246.8
1,032.2

947.0

876.0

EDI : ; conj.
:enhonol
967.6 29.98
311.6 18.74 no hydro-
897.6 87.60 quinone
636.9 28.95
s ' ‘
340.9 43.87
496.5 38.09 no hydro-
316.3 23.92 quincne
371.04 28.22
341.7 81.63
1; . .
818.9 37.30
1,364.6 38.72 1 gs. hydro-
l,1ll.08 40. 98 quinone daily
396.3 83.19
1126406 81.92
1’ O
1,081.6 39.004
930.7 43.08 1 gm. hydro-
l,147.d 36.18 quinonc daily
1,661.1 17.88
752.7 19.86
.4‘ .
2.499e8 45e05
‘1’e5 66.81 2 me hydro-
l,863.5 44.22 quinons daily
1,468.9 34.82
2,160.0 86.51
a, . .
1,774.9 48.41
2,626.08 68.6 3 gas. hydro-
8,378.1 64.63 quinone daily
1,883.7 49.72
1 890.7 53.63

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Juno 3
June 6
June 6
June 7

June 9
June 10
June ‘ 11

June 16
June 17
June 18
Juno 19

1st week
2nd week

6rd weok
4th week

6th week
6th week
7th week

8th week
9th week

 

 

 

 

fie phonoI 56531 'p'EcnoI : % conj. *
per day; : 2r dg : Ecnol W
1,680.6 1,844.6 14.61
‘56s? 732s: 40.37 no hydro-
666.6 1,069.6 68.71 quinone
641.2 868.7 67.70
695.4 1,140.4 47.96
. 0‘ Q 0 0
917.9 1,602.7 29.62
401.2 666.08 29.11
636.2 840.8 24.44
664.4 661.8 14.94
624.6 640.8 18.17
p 9 ‘, e e
601.2 776.7 22.60
662.4 744.6 24.47
674.6 711.7 19.28
626.00 692.6 24.20
, e , e e
1,966.9 2,706.9 27.66) no hydro-
6,170.4 4,964.6 26.04) 1 gm.
6,907.1 6,676.4 29.66) 6811’
6,046.6 8,011.8 67.02) 2 El. 6811!
6,067.0 10,642.6 61.96) 6 gm. 1811!
3,808.4 6,666.8 32.6 ) no hydro-
5. 033. 2 4, 002. 2 24c 21) “man.
2,266.17 2,922.8 22.41)

12

In the case of dnimal II, the same experimental
procedure was followed as with Animal I, with the
exception that creatinine was also determined daily.
the results, howcver, were somewhat different as say be
secn.in.table II.

In this case there was a marked increase in the
conjugation of urinary phenols as soon as the amninistration
of hydroquinone was started. the percent of conjugation
did not increase further, however, when the amount of
hydroquinone given was increased. With.inimal 11, using
the two week control period.as a basis for calculation,
approximately 86% of the hydroquinone fed was excreted
as free or conjugated ”phenol“. is soon as the feeding
of hydroquinone was stopped the urinary phenols returned
at once to normal values and the percent of conjugation
dropped to the values obtained before the administration
of hydroquinone.

the creatinine excretion resained fairly constant
throughout the experiment. the pig seemed to develop
normally and no deleterious effects of the experiment could
be noticed. this annual was approximately the cane sise

as the preceding one.

16

 

 

 

22883 11
Inc phenom phenol: i conju-. . 2
per Q per g :gation :creatinine:
06‘s 21 464.4 695s: 66.2 997a:
Oct. 22 882.4 780.8 12.8 1,088.0 no
Oct. 28 888.1 766.3 28.1 981.8 hydro-
Oct. 24 828.9 887.8 21.8 1 029.8 quinone
I.“ 5.5 2,663.3 2503 1.0g501
Oct. 27 We“ 496.6 11.2 1,901.,
Oct. 28 788.8 829.8 7.8 1,080.8 no
Oct. 29 819.2 681.8 4.9 1,061.7 hydro-
36:. :10 2153.‘ gage: 1:0: 999e9 qmou ‘
o e 8 e . . e e . 908.06
tfi 4 2,372.. 16'! 6,601.66
[07. 3 808.4 1,007.02 19.9 1,208.1
10's ‘ me? 866.1 61.4 1,158e7 1 no
lov. 8 878.8 924.1 87.7 1,817.8 daily
889. 8 881.8 989.8 48.1 1,009.3
Nov. 7 42.7- 841.02
lot. 10 774.9 1 078.8 27.9 1,8 9.
‘Ove 11 619.1 .886.1 60.1 1.1:)»: 1 8..
. . . 17. .
luv. 14 818.7 3 1’168 1
9
IOYe 17 680.1 866 9 c
10'. 18 688.7 896:3 11:35 1'193.: 1 ga
luv. 19 844.. 988.8 89.8 1'042.s dail.
nov._20 889.4 989.08 81.1 1:116.6 y
101'. 21 $80.9 1,063.9 £6.00! 1,818.9
1.7. 2. 818.8 , 1:431:l 48.8 ' I
10'. 28 890.08 1.088.007 42:07 1’3::.: 2 gms
rev. 28 889.1 728.2 48.19 '879.9 dail °
10?. 27 980.8 1,840.7 80.8 1.288.7 ’
not. 28 827.7 801.1 84.88 ’--..

 

 

    

   
 

 

 

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fie phenol .7372: iEenoI 3 confu- :
Er day Er day gationg Oreatinine:
DOC. 1 861.8 1,269e7 3‘e48 1.30808 ‘
300. 2 724.8 1,048.1 60.8 974.7 2 8212.
D00. 3 884.6 1,237.6 28.6 1, $2.06 48.11,
DOOe ‘ 798.5 1,019e03 21.1 878e5
mac 5 7‘3e1 771.06 2.6 1 031.6
. O . 0 O ’ 0
"Ce 8 649.8 826.6 ”e‘ 1,020o‘
n... 9 1,081.0 1,821.8 88.88 --- a? .
DOB. 10 861.7 1,261.9 64.8 cum 1’
“Os 11 729.2 1.079s? 62.4 ’19e8
DUO. 12 694.7 967.4 27.4 ---
m4 , . .
DOC. 15 498.1 490e5 105 '"""
“Co 16 649.2 412.7 1‘.‘ -"' no
D00. 17 244.4 661.1 4.6 hydro-
Dec. 18 417.6 423.4 1.4 quinone
D00. 19 270.6 686.9 60.07
. O . 0
no
1.2 '00: 2,235.8 2,908.8 ”e3 4,096e1) ”‘30-.
2nd week 2,801.4 2,872.9 10.8 8,901.88) quinone
m '00! 2, 908.2 4,456.5 64.9 6, 829.92)
4th week 8,089.8 8,891.1 27.7 8,298.8 ; 1 an.
an '“t 3.“? 2, 339.‘ "e5 6,176.0 6811
56h 'OCk 2,264.7 5.35‘e1 69.6 5. “9e' )
7211 I002 5,982.8 6,346.6 26.7 6,296.6 ) 2 “I.
8th week 6,886.4 6,146.9 68.6 on ) daily
921! I002 1,879.8 2,076.7 10. 67 --- IO
hydro-

15

The experiment wee repented 8 third time,
using the one experimented mind on in the preceding
oeee. rho reeulte of thie third triel, en given in
hble III, perellel thoee of the eeeond to e feir degree.
Upon feeding hydroquinone there wee en inediete inereeee
in the excretion of both free end eonjugeted phenols.
the peroent of eenjmtion wee eleo euhetentiell: in-
ereeeed. n in the preeeding triel there wee no further
inoreeee in the peroent of oonjugetion upon inereeein‘
the mount of hydroeuinone edninietered. lhen l p.
wee fed deil: ehout 88$ of the'hydrequinone wee exereted
ee free or oonjugeted 'phenol', end when 8 no. were
fed an: eheut 98$ 9... excreted. Upon the eeeeetion of
eduinieterin hydroquinene the free phenol, totel phenol
end percent of e0n:u¢etion inedietel: dropped to
epproxinetely the one veluee ee before the experiment.
In eontreet to the preceding experiment, however, there
eeened in fliie oeee to be e definite greduel inereeee in
the ereetinine excretion upon feeding hrdrequimne.
hie reeohed e peek during the fifth week end frol- then
on greduelly returned to e nor-e1 velue.

2125! III

88 ene:

 

Jen.

3800'

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Jule

Jun.
:80.
:83.
:me
38110

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Jen.
Jen.
Jun.

’06.
’O8e
’06.
1.60
200.

”he
’000
1.6.
7000
1.6.

’86.
’86.
’06.
’08 e
’06.

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16

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388813

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16

 

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r Etion :creetinine:

626.00 727.9 14.4 1,088.06 no
682.28 794.0 26.6 1,297.6 614300
406.8 647.6 26.8 1,124.6 1316660
446. 9 584.. 8 24. 8 1, 850. 7
621.2 404.6 20.6 1 206.4

W‘ ’ 0 O Q 0
489.2 696.9 17.62 1,644.9 60
400.1 480. 6 10. 7‘ 1,1180. ””0.
8060 5 2130 05 110“ 1,167e3 {8.150110
667.2 406.8 17.11 1,020.9
282.8 668.7 21.14 1,269.8
’ O ’ O ’ 0
668.6 678.7 68.06 1,021.89
676. 7 85808 68.47 1. 684. ‘1 1 “e
686.9 920.6 66.66 1,616.2 4811’
4.78.8 877.8 . 29.81 1,881.7
667 9 - 969.18 2.72 1 679.

W 1.8M . , .
469.9 789.6 41.22 1,262.9
468.2 696.6 61.97 1,290.08 1 GI.
692.0 1,197.9 42.24 1,966.4 4011]
700.7 1,044.6 62.9 1,686.6
626.0 606.6 66.11 1 086.6.
. e ‘.uze' “0’ 7,1760;
888.8 1,088.4 88.9. 1,807.8
078. 7 1,113. 8 '9e06 1, 6040 6 1 ’0
666.6 972.06 64.82 1,618.6 4311!
672.2 1,066.6 66.66 1,680.7
666.7 1,010.6 66.97 1,664.0
, O . 0 0 ’ 0
689.6 1,206.06 42.81 1,480.2
680.1 1,068.09 46.69 1,096.6 2 .
601.2 1,087.66 44.8 1,669.2 6 1’
880.8 1,108.00 41.8? 1,084.8

759.3 1,173.40 66.28 1,076.;
. 0 ’ 0 0 . 0

17

m III (Can't)

 

 

 

 

 

 

EC. 556661356: P5830: 3 I OOflIn-DS f
2.; as! i 21' g : £88106 30266816186:
1.8. 24 622.7 1,104.6 46.62 1,167.8
lab. 26 709.7 1,269.6 42.76 1,667.08 2 m.
166. 26 - 661.1 969.7 41.64 1,268.6 6611’
BB. 27 466.7 869.2 66.79 1,666.6
206. 28 706.4 1,069.8 64.61 1,219.2
~ 9 ° 9 ' ° 9 °
m. 6 491.82 946.88 47. 86 1,067.8
11.8. 4 668.88 1,048.58 88.21 1,898.6 8 an.
m. 6 617.94 1,046. 78 40.80 1,166.0 8611’
m’ 3 :38? 12:2"? 8'3: ,1: 188%.
m. . . 0 .
2,902.5 5,090.6 .01 , .
m. 10 464.6 668.08 16.66 1,090.6
In. 12 468.6 671.02 26.01 1,218.6 hydro-
mo 13 36605 48905 2505' 1,106.4 awn.
m. 14 479.1 679.1 29.66 1 149.6
. 6 , 6 O . 6
hr. 17 474.8 661.4 16.28 869.6
hr. 18 464.4 626.6 27.11 1,169.07 118
hr. 19 606.6 706.2 28.62 1,221.40 hydro-
m. 80 875.0 495.8 84.31 1,121.4 111111011.
118:. 21 69. 600.9 26.91 1 096.09
. 6 , O 0 , 0
1.8 1...: 2,882.4 8,088.7 88.08 6,947.2) no hydro-
2110. .802 1,876.1 2,266.2 16.7 6,877.6) 11111100.
8178. Ink 8,888.8 4,026.1 88.8 8,888.8) 1 an.
4th an]: 2,616.9 4,263.9 40.9 7,176.4) 0.6117
6th not 6, 287.8 6, 207.9 66.8 7,446.2)
6th um: 8,880.8 8,848.9 41.9 8,099.1
70: no.1: 8,060.4 8,888.7 89.8 8,878.1 2 gm.
8811 tuck 2,902.6 6,090.6 46.01 6,811.6) 6.8.11]
98:: no.1: 8,179.0 8,887.0 84.8 6,447.4) no hydro.

1081: Ink 8,179.0 8,887.0 24.3 6,447.4.) 011111011.

18

In repeating the experiment a fom'th time a new
experimental animal was obtained. It was impossible to
obtain an animal as large as the two preceding ones had
been. Animal III, weighed about 40 pounds at the be-
ginning of the experiment. iswell as repeating the
previous work. inorganic sulfates, total sulfates and
total nitrogen were determined.

The pig was growing rapidly during the course
of the experiment. this would bring about a normal
increase in all the substances determined which must be
taken into consideration in analysing the data. {the
data obtained in this fourth experiment (hble IV)
differs quite radically from those of the three preceding
ones. Upon feeding hydroquinone there was an increase in
urinary phenols, but the increase was almost entirely
free phenol. fhe data show that conjugation was almost
negligible during all except the last two weeks of the
period in which hydroquinone was fed. Detwun the period
in which 1 gm. of hydroquinone was fed daily and that in
which n gms. were fed daily no hydroquinone was given for
s week. During this week urinary phenol fell back
inediately to normal values, although there was absolutely
no conjugation. The only explanation that can be offered
for this absence of conjugation is that the capacity of a
young rapidly growing animal for conjugation is probably
very limited and needs to be developed.

19

The first week of the period in which two grams
of hydrcquinone were administered daily shows an ab-
normally large increase in phenol excretion. Using the
preceding week, in which no hydroquinone was given, as a
basis, there was practically complete elimination of
the hydroquinone during this first week on two grams
daily. the second and third weeks of this period. or
the eighth and ninth weeks of the experiment, show
values for phenol excretion more nearly parallel to
those earlier in the experiment.

. During the eighth and ninth weeks there be-
gan to be some conjugation of the phenols. this con-
jugation steadily increased to the end of the experiment,
no decrease being shown when the administration of
hydroquinons stopped. this might substantiate the idea
that lack of conjugation earlier in the experiment was
in some way connected with the age of the animal. low
that the animal had grown older, conjugation of phenols
increased.

”During the period when one gram of hydro-
quincne was fed daily approximately 80% of the hydro-
quinone fed was excreted as free phenol. During the
period when two grams were admihistered daily,
approximately 85% was excreted. this was also almost
entirely free phenol.

80

as was to be expected there was a gradual
increase in creatinine output during the course of the
experiment. Apparently, however, when hydroquinone
was first fed there was an abnormal increase in
creatinine output. rrom this time on, the increase was
again very gradual until the feeding of hydroquinone
was stopped, when there was a slight decrease. rhe
hydroquinone apparently had a definitely stimulating
effect on creatinine elimination in Animal III.

rho data obtained from the determination of
inorganic and total sulfates are rather inconsistent.
there was a gradual increase in both during the course
of the eneriment. This increase did not seem to be
affected by the administration of hydrcquinone and was
probablydue to the nomal growth of the animal. Although
the values obtained for percent of conjugation of sulfates were
inconsistent, a definite increase in ethereal sulfates dur-
ing the feeding of hydroquinone is indicated.

The data on total nitrogen are also somewhat
difficult to detemine. There was a definite increase in
total nitrogen elimination when hydroquinone was first
fed. During the three weeks in which 1 gm. of hydro-
quinone was given daily, the total nitrogen values re-
turned te approximately normal, however. When the feeding
of hydroquinone was discontinued for a week, total nitro-

gen excretion decreased enormously to a value about half

£1

that of the normal. When the feeding of hydroquinone was
reamed and s gms. were fed daily there was a great in-
crease in total nitrogen output to a value about four
times that of the preceding week. The total nitrogen
values then continued to be high as caspared with the

two weeks control period, but they decreased gradually
during the three weeks that a gas. were given daily.
After stopping the feeding of hydroquinone, however, in-
stead of dropping as in the sixth week, the total nitro-
gen values increased substantially.

Ihese data seem to show that the total nitro-
gen was definitely increased in animal III upon feeding
hydroquinone, but that some sort of adjustment was made
upon continuati on of administration of hydro quinone
which permitted the values to return to normalcy.

The figures for creatinine and total nitrogen
both seem to indicate that hydroquincne caused a certain
amount of tissue destruction in animal III.

 

 

 

 

 

 

 

 

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on no.H coma. Oman. n.ewm m.om m.¢om m.nmm mm .nn4
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« now «
«soapfizu .madm « .Madm ccfimdpwcnuu noapww had non had non
« Hence“ Hence « cancwaonH ulfimmoo R“ Hoccnm proe Hmnonm comm

 

>H mgm<e

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r.

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oHueH, aw.HH no.0 m.>mHaw mo.Hm m.Hnm.m m.oomMH
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-Hsu w>.H mem>.m mmnm.m m.Hmm mm.am H.mme o.mmn n aHeh
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on mH.e m>n¢.m momm.m «.mem mo.Hm m.wmm m.mne H aHsn
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.sw m om.H mman.m Hmmo.H m.m>o mm.mn n.m>e.H m.m>m mm ones
mn.m omem.H oHa¢.H a.a¢o.H om.mm e.omn.H n.mHo.H em mesh
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[8.3 3.3 mmH.m lama}. Rmfi 0.:
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mm.m banm.H amem. o.mom mm.wm ¢.¢mo.H o.mmm mH ouch
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.em m om.m mbwm.m omHm.H m.¢om mn.>H m.meH.H m.mwm pH ones
moem.H amom.m moHH.m m.¢ma --- o.moa.H o.mmb.H mH ouch
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u new « u u u «
«nonpazu .mazm « .mafim « onHmauaonun mafiuww u had non u had non
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Auafloov >H mamde,

 

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we mom mam men RHN mmm man Rm RaH RQH mo mopaaHsm
.mfinnoo R
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on AoH.eH ee.HH bo.m m.anH.¢ mo.Hm m.Hnn.m m.oow.H ace: QQOH
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page N AnH.eH mo.m Hm.» a.mmH.n mm. >.m¢w.mH m.mmm.mH some nab
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.am H A-- mm.» nn.o m.Hee.m a.H n.0He.m m.m¢w.m noes can
OHHOHH
lamb
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0: nom.o mm.e om.e n.omm.H w.mH m.nmm.H b.mnn.H Moo; pmH
ImH.mH, Hmm.HHe -mm«oH, m.nHH»¢ m.mm ‘n«.mm>.m ..mom.m
0:02 ec.m mwmm.m mwmn.m m.mma na.mm w.omo m.ew¢ HH aHsn
sHsu mm.» onmo.m onmo.m n..Hm oo.>m >.moe o.me 0H chn
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on am.n «HOH.N emom.H m.nom po.mm m.>mo H.mm¢ m chh
mo.e momm.m onee.m n.mma n.mH >.nmm b.0ae a aHeh
« new.“ a u m, w. 4 - “-
«Ioanzu.mHSm « .MHSm « onfianconou GOprm « hen mom « had mom
I, “ Hence" Hmpoa “ cancwnonHu «tahaoo-Nm Honogn fiance“ Homonm comm

Aw.pnoov>H mgmne

25

for the fifth experiment another animal was
obtained. this animal weighed about 60 pounds, at the
beginning of the experiment. The data are shown in
table U for free and total phenols, total sulfate and
total nitrogen. It will be seen that these data are
very admilar to those of the preceding experiments.
Upon adunisterim hydrequinone there was an increase
of both free and conjugated phenols, but little change
in the percent of conjugation. About 49$ of the hydro-
quincne was excreted as urinary 'phenol'. the total
aunt. output was lowered slightly a: the feeding of
hydrcquinone, the daily value of total sulfate averaging
.38 gms. less than during the control period. this
decrease in total sulfur elimination was more pronounced
in the succeeding experiment (fable 71) but is also
suggested by the data of experiment 1'.

   
 

con u—:

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gene; ; “tics 0,. Sul glitroggg

 

_, 1,
Apr. 7 487.9
Apr. 8 818.1
“to . “£03
”to 10 586s 5
Apr. 11 425. 7
”to 1: ‘79e 2
Apr. 13 666.8
Apr. 1‘ $1. 08

e 0 e
we 17 531s 5
Apr. 18 708.8
13!. 19 1,041.6 1,
Apr. m 1, 134.0 1,
”1‘s 21 1, 088. t 1,
”1‘s 22 1, 075. 8 1,
Apr. so 1,039.9 1,
Apr. 2‘ 78‘e9

0

667.2
449.2
669.2

696.02

494.1

409.06

741.6
823.6

0

847.4
940.6
071.6
195.9
137.4
267.8
237.9
964.1

a

12.44
29.19

8.24
16.56
14.27

7.28
10.21
69.19

26.6
24.66
65.53
6.02
4.74
14.47
16.00
18.68

2.22
1.92
1.61

I.‘ ' 2e19

2.68
1.48
8.91
2.21

2.46
2.68
2.26
2.64
1.91
2.4

2.00
1.96

O

4.54

5.69

‘e” 80 hydro-
6.71 (“130“
6.58

8.46

6.57

4.45

9

6.60

5.44

6e11 1 as
6.26 um-
5.0e quinoae
5.88

6.82

7.02

27

rho ministration of hydroquinone sening to
have no serious effects on the experimental animals
used, the author undertook a similar experiment using
himself as the subject. In this experiment inorganic
sulfate, total sulfate and total nitrogen determinations
were detemined, as given in fable '1. lo attempt
was made to control the diet during this experiment
and this must be borne in mind in comparing the data
with the previous experiments. Upon taking hydro-
quinone there was a very definite increase in conju-
gation of sulfates, about a 35% increase. the total
sulfates showed a decrease which averaged about 1 gm.
per day, while hydroqninone was being taken. Since the
diet was not controlled the true significance of these
values cannot be determined. l'here was an mediate
definite increase in total nitrogen upon taking hydro-
quinone, the values soon returned to normal, however.
The phenol deteminations also were similar to those of
of previous eneriments, there being an increase in
both free and conjugated phenols but no change in the
percent of conjugation. About 66% of the hydroquinone
appeared as urinary "phenol”.

 

 

 

 

 

 

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nm.aH mmH.¢ nme.n HH .nwz
am.em can.» nmm.m 0H .cez
om.om mnm.¢ aom.m m .nnz
mH.HH mHm.e mam.e m .cez
n¢.mH ama.n oeH.e m .nez
m©.mH mmo.m mHm.e a .nag
am.nH mmm.m mmw.m n .nea
am.nH mHm.m .one.e m .nwa
mm.eH mHo.m mHH.m H .cnz
mm.m mn.m ©.mm o.>OH.H ¢.mHo.H pH.uah
.mnwoo -zWo.mH m.¢mm.H m.m>m.m m.weo.m
om.m m.> H.©w ®.meH.H e.mmoww,mH.eam
mm.mH m.nH >.mom m.mm¢.H m.omm.H mH.neh
OH.HH H.mn n.nne m.eon.H m.wa eH.nws
om.0H H.nn w.noe n.pHm.H m.mHm nH.eeh
HH.>H ¢.m n.mOH m.>eH.H «.mno.H NH.nen
mm.o© me.om m.nwn >.Hmm.m o.m>m.H
om.mH m.on H.wwH m.mmm a.>mn m .cah
mm.HH m.m m.mm wo.oe¢ m.eH. m .sea
mo.mH m.Hm m.mmH m.mmm m.am¢ a .nwn
mm.mH m.mH p.05 n.0me o.oan o .nah
ma.OH m.mm m.w0H o.mon oo.mmm :m .nan
u 93an «onmwasmu cpwmgm « .z u noapnw «Johanna u Hoccnmu Hosann-
u ufiwmoo R ... flnfioeuonmmnwmoshu Hmuroe « 33.500 R «Hmoaoflpm u Halves“ comm

 

H> mqmdu.

29

Isolation of Urinary Compounds
Attempts were made to isolate hydroquinone
and muconic acid from the urine of animals receiving
hydroguindne. For the isolation of hydroquinone the
method of Baumann and Preusse (15) was used. The urine

was heated after being made distinctly acid with H01. It

was then thoroughly extracted with ether. The ether ex-
tract was evaporated to dryness and the residue dissolved
in water and neutralized with barium carbonate. This
solution was then again extracted with ether until the
water solution would no longer reduce Tollen's reagent in
the cold. The ether extract was evaporated nearly to dry-
ness and crystals of hydroquinone appeared. They were
purified to some extent by recrystallization from toluene.
However, the author was unable to entirely free the
crystals from the pigment extracted with them.

In this manner crystals were obtained melting
at 138‘3 - 140°. The crystals sublimed without decompo-
sition when heated with ferric chloride; dissolved in
ammonia yielding a brownish liquid; sublimed, when heated
rapidly in an Open test tube, giving an indigo blue
color, all of these prOperties corresponding to those
of hydroquinone. Because the crystals could not be
completely freed from pigment, their weight could not be

accurately determined. Colorimetric analysis, however,

 

 

chewed the ether extract of a two day urine a-ple of
an animal receiving 1 an. or hrdroqninone daily to
contain .0286 53a. of phenolic enbatanee.

Several attenpta were made to iaolate muconic
acid nains the method or leunaerker (a). the urine
anple waa evaporated to a amp and extracted for
twelve honre with ethyl acetate. rhe ethyl acetate
waa extracted by ehating with eaturated eodinn carbonate
until no more carbon dioxide wae evolved. the eodin
carbonate solution wee heated until the odor of ethyl
acetate could no longer be detected. The solution wee
than nentraliaed with n 01 to conga blue. laconic
acid, if present, ehonld have precipitated at this
point. However, no muconic acid wee found in tour
attanpte.

The hydroqninone that wae not excreted aa a
phenol, it oxidised, wne probably carried beyond the
atege of mania acid.

Although there are many variations in the
six experiments, due probably to differences in
experimental animals, several facts stand out concern-
ing the fate of hydroquincne in the animal body. In
each case upon administering hydrequincne there was
an immediate increase in both free and conjugated
phenols in the urine. The percent of conjugation was
changed but little, however, unless the mount of hydro-
quincne given was increased to at least 2 gas. daily.
fhe percent of conjugation was then increased, the
amount of the increase varying with individuals. The
percent of the hydrcquinone excreted as fed varied from:
25% to 80%. At least a part of the hydroquinone was
excreted in either the free or conjugated fora without
having been changed by passage through the body. 1'he
portion of hydroquinone unaccounted for by the urinary
phenols may have been oxidised past the stage of muconic
acid, presuably to carbon dioxide and water.

The total sulfate excretion was lowered by the
feeding of hydroquincne. rho decrease was most prominent
in the last case (see table VI). the exact significance
of this is open to discussion. It may be sugeated,
however, that Bhiple, Huldocn and Sherwin (10) found
total sulfates to be lowered when cystine was fed with
0535 Br, due to the excretion of 0635 hr. as a

meroapturic acid. The percentage of conjugation of
sulfates was increased substantially in each case.

The excretion of total nitrogen and creatinine
were both definitely stimulated upon administering hydro-
quinone. The values for both of these substances tend-
ed to regulate thanselves toward the normal, however.
This would suggest that hydrcquincne causes an increase
in the catabolism of body tissues, but that the eniael
body has a tendency to adjust itself to eliminate this
extra tissue destruction.

These conclusions may be smarised briefly
as follows:

The feeding of hydrcquinone brings about,

1. Immediate increase in urinary phenole,

8. Little or no increase in the percent of
conjugation of urinary phenols,

5. A slight decrease in total sulfate values,

4. A definite increase in ethereal sulfates,

5. A definite stimulation of creatinine and
total nitrogen excretion, both of which
tend to return to nomalcy, however.

Bydroquincne, but no muconic acid, can be

isolated from the urine of animals receiving 1 gm. of
hydrcquinone daily. . a

'D

10.
ll.
12.

BIBLIOGRAPHY

Huston, Lightbody and Ball, J. Biol. Chem. 79, 507-18.
Husa and Russ, J. Am. Pharm. Assoc. 17, 243-7.
Neumaerker, Ztschr. fur physiol Chemie, 126, 203.
Fuchs and v. Sdos, Ztschr. fur physiol. Chemie, 98, 11-13.
Underhill and Harris, Jour. Ind. Hyg. 4, 491-500.
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