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Some observations on urobilin output in sprue
290
SOME OBSERVATIONS ON U R O B I L I N O U T P U T IN SPRUE. BY R. M. MORRIS, M.D. (Lond.), D.T.M. & H. ~
Salisbury, Southern Rhodesia.
I.--INTRODUCTION.
Our knowledge of urobilin dates from 1868, when JAFFE (1869) first discovered its presence in some urines. MALY (1871) obtained a product-hydrobilirubin--by a direct reduction of the bilirubin in bile, and was able to demonstrate its identity with JAFFE'S urobilin and the stercobilin, which VANLAIR and !V[ASIUS (1871) had procured from f~eces. HOPPE-SEYLER (1891) was one of the first to emphasize that in urobilin one had to deal with the normal, final, product of bile-pigments, which, in turn, were derived from the blood-pigment, haemoglobin. This point was utilized by EPPINgER (1920)', who sought in this way to obtain a quantitative estimate of blood destruction. In sprue, the interest in this substance is two-fold. Firstly, the peculiar and characteristic colour of the freshly-voided faeces in this disease excites interest in any matter which concerns stool-pigments, and secondly, the anaemia accompanying the severer cases is worthy of deep and continued study. Hitherto, whilst the qualitative tests for urobilin in urine or faeces have been a simple matter, the quantitative ones have involved laborious processes, with special apparatus ; but recently TERWEN (1924) introduced a simple colorimetric method, which insures reliable results and is easily applicable in an ordinarily-equipped laboratory. It is with the aid of this method that the data in this paper have becn obtained. II.--PROPERTIES OF UROBILIN AND UROBILINOGEN.
Before proceeding further, it may not be out of place to recall some of the properties of these substances. JAFFE described how solutions of zinc salts of urobilin, in alcohol or chloroform, present a remarkable fluorescence, which appears pink by transmitted and green by reflected light ; on which property is based the usual Schlesinger -e Thesis approved for the Degree of Doctor of Medicine in the University of London. From the Hospital for Tropical Diseases, I.ondon, and the Department of Pathology, London School of Hygiene and Tropical Medicine.
SOME
OBSERVATIONS
ON
UROBILIN
OUTPUT
IN
SPRUE
291
test for urobilinuria. The other main test is the absorption band in the greenblue area of the spectrum--the exact position varying slightly according to the reaction and nature of the solvent. The zinc salts also have a characteristic band in the same area. Urobilin itself is a labile substance, which hitherto has been prepared only as an amorphous, reddish-brown powder of uncertain purity, and it has been shown that the above criteria are group reactions, not specific; hence, chemically, urobilin may be of several varieties; clinically, however, this is of no moment. Another point noted by JAFEEwas that, in the urines which gave the fluorescent test, the dark colour did not appear till they had been standing for some time ; he, therefore, postulated a colourless chromogen. Later, this substance was isolated from urine and named urobilinogen. It has been prepared in crystalline form and shown to be a reduction product of urobilin, into which it returns under influence of light and air (SAILLET,1897). The relationship may be shown graphically thus :-Bilirubin-----~- Urobilin ~ Urobilinogen. The main test for urobilinogen is the Ehrlich's aldehyde reaction, where a deep red colour is imparted to the urine by the addition of a few drops of 3 per cent. paradimethylamino-benzaldehyde in 50 per cent. hydrochloric acid, which red colour presents an absorption band in the green. A second test is a diazo-reaction, where Ehrlich's diazo-reagents produce an orange colour in the urine, changing to a citron-yellow on adding ammonia. These reactions again are " group reactions," but for clinical purposes suffice. III.--METHODS
EMPLOYED.
URINE.--All the tests were carried out on 24-hours' specimens of urine collected in a black bottle. As a routine, Ehrlich's aldehyde reaction for urobilinogen and Schlesinger's test for urobilin were performed directly the 24hours' specimen was received. If positive, the total quantity was measured and a portion set aside for quantitative examination, in which no attempt was made to differentiate the proportions of urobilin and urobilinogen. F~EcES.--These estimations were also carried out on 24-hours' specimens, collected from noon to noon and kept in an opaque jar, with a tightly-fitting lid, and in a dark corner. The total quantity was then weighed and the whole mass rendered as homogenous as possible with a pestle. Two portions of 5 g. each were then weighed out as rapidly as possible and the estimations carried out on them. One portion was used for the estimation of the quantity of urobilinogen and the other for the total urobilin plus urobilinogen present. Each patient's excreta were examined daily for 7 days. TE~WEN'S method is a colorimetrie one in which, first, all the urobilin present is reduced to urobilinogen, the latter extracted in a suitable solvent, and the aldehyde reaction performed with this solution. The intensity of the colour developed is then
29~
SOME OBSERVATIONS ON UROBILIN OUTPUT I N SPRUE
compared with that of a standard, alkaline, phenolphthalein solution in a colorimeter, and this result expressed in units of concentration. Gravimetrically, TERWEN has shown one unit of concentration to be equal to 0"4 mg. urobilin per 100 c.cm. urine or 100 g. f~eces. If the reduction step in the process be omitted and a selective solvent, e.g'., petroleum ether, which extracts urobilinogen but not urobilin, be used, a quantitative estimate of the amount of the chromogen alone can be obtained. DETAILS OF PROCEDURE.
THE F ~ c E s . - - T h e 5 g. of f~eces are made up into a homogeneous emulsion with 50 c.cm. of distilled water and 50 c.cm. of a freshly prepared 16 per cent. Mohr's salt (FeSO4, (NH4)2SO4, 6H20 ) solution added. Then, in a fine stream from a pipette, 50 c.cm. of 12 per cent. solution of caustic soda are allowed to run in, the beaker being constantly agitated. This results in a greenish, turbid liquid, which is forthwith poured into a flask so that a glass stopper can be inserted without the presence of an air bubble. The whole is then put away in a dark cupboard for at least eighteen hours, during which time the urobilin should be reduced to urobilinogen. That this has duly taken place must be controlled by the spectroscope, when the urobilin band will be absent if reduction has been complete. The fluid is then filtered through a pure, dry filter into a brown flask. The rest of the process must then be carried out without interruption, as the urobilinogen tends to pass into urobilin by the oxidising action of air and light. 5 c.cm of the filtrate are then accurately measured and placed in a clean, dry, separating funnel fitted with a stopper, in which it must be rendered acid by the addition of 2"5 c.cm. of a 20 per cent. tartaric acid solution. (This may be controlled with litmus paper). Into the separating funnel is then measured 40 c.cm. of petroleum ether (s.g., 0"65 ; boiling point 40 ° to 60 ° C.) and the whole shaken up about seventy times. On separation of the layers, the lower may be allowed to run off, as the urobilinogen will have passed into the petrolether. From this latter, 20 c.cm. are measured off into a second, clean, dry, separating funnel, and 20 c.cm. of ether added. (This ether must be p u r e - commercially pure ether washed with caustic soda and distilled water and then distilled.) To the mixture are added 5 c.cm. of a saturated solution of paradimethylamino-benzaldehyde (MERci() in purified ether. Rapidly after this 0"5 c.cm. of 38 per cent. hydrochloric acid is added and the whole vigorously shaken for one and a quarter minutes, when a few c.cm. of water--recently distilled--are blown in from a wash bottle. The shaking is continued for another fifteen seconds, and then 3 c.cm. of a saturated solution of sodium acetate are added. This causes a milky appearance, which disappears on further vigorous shaking. The purpose of the acetate is to remove the colour reactions due to indol and skatol bodies, which, having unsaturated
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
298
pyrrol-rings, also give red or reddish-blue colours with Ehrlich's reagent (e.g., the cholera-red reaction). The now clear, reddish-blue fluid in the lower layer is allowed carefully to run out into a measuring flask. A further portion of urobilinogen can be made to react by repeating the process from the addition of the hydrochloric acid, and the resulting fluid is added to that previously obtained. The quantity is then measured and the fluid is used in a colorimeter. TERWEN uses an Authenrieth colorimeter, for which a curve is plotted, but nearly as accurate results may be obtained by using a Dubosc instrument and giving the result as a percentage of one unit of concentration, one unit being defined as the concentration which gives a colour, in an " end-fluid " of 10 c.cm., exactly matching the colour of the standard. This standard, which must be freshly prepared each day, consists of 5 c.cm. of a 19 per cent. anhydrous sodium carbonate solution (saturated), 1 e.cm. of a 0"05 per cent. solution of phenolphthalein in alcohol, and 94 c.cm. of freshly distilled water. Further, in calculating the result, the progressive dilutions must be taken into account, vi~. :-1. From f~eces to reduction fluid 5 to 150 -- 30 × 2. From filtrate to petrol-ether solution, 5 to 40 = 8 × 3. From petrol-ether solution to " end fluid." 20 to R. and the percentage reading must be multiplied by these. This gives the result in units of concentration, from which the weight in milligrammes of urobilin in the stools can be obtained thus :-W × U × 0"4 = mg. of urobilin per diem ; xv~ w h e r e W = w e i g h t of 24 h o u r s ' f~eces in g r a m m e s , U -- u n i t s of c o n c e n t r a t i o n pre s e nt .
The reaction for the quantity of urobilinogen present is performed in exactly the same way, except that the emulsion of the 5 g. of f~eces is made up to 150 c.cm. with distilled water and the reduction process is omitted, the diluted f~eces being immediately filtered. The result is expressed as urobilinogen in terms of mg. of urobilin. THE URINE.--The Qualitative Tests.
Urobilin--Schlesinger's Test.--20 c.cm. of urine are acidulated with acetic acid and the urobilin extracted by gently inverting with 5 c.cm. of amylic alcohol. This layer is then pipetted into a second clean, dry, test-tube and a few drops of 10 per cent. alcoholic solution of zinc chloride added. If positive, a green fluorescence develops and an absorption band in the green-blue may be seen with a hand splectroscope. This indicates an excess of urobilin. Urobilinogen--Ehrlich's Aldehyde Reaction.--To 5 c.cm. of urine 2 drops of a 3 per cent. solution of paradimethylamino-benzaldehyde in 50 per cent. hydrochloric acid are added. A deep red colour gradually makes its appearance,
"29~
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
more readily if warmed slightly. In this also can an absorption I t is p o s i t i v e w i t h a n e x c e s s o f u r o b i l i n o g e n . Quantitative
band
be s e e n .
Test.
N o a t t e m p t w a s m a d e to d i f f e r e n t i a t e t h e t w o s u b s t a n c e s . The process is v e r y s i m i l a r to t h a t o f t h e f~eces, b u t 8 0 c . c m . o f u r i n e , f r o m a w e l l - s h a k e n 2 4 - h o t l r s ' s p e c i m e n , a r e u s e d , a n d t h e r e d u c t i o n c a r r i e d o u t w i t h 20 c . c m . of t h e M o h r ' s salt s o l u t i o n a n d 20 c . c m . c a u s t i c s o d a . A g a i n it m u s t b e l e f t in t h e d a r k , w i t h all air e x c l u d e d , f o r at l e a s t e i g h t e e n h o u r s . From the filtrate, 20 c.cm. are taken and acidulated with 5 c.cm. tartaric acid and the extraction performed with 80 c.cm. of petroleum ether, of which 60 c . c m . a r e u s e d i n t h e s e c o n d s e p a r a t i n g f u n n e l . T h e q u a n t i t y o f e t h e r a d d e d is 60 c . c m . a n d 5 c . c m . o f E h r l i c h ' s a l d e h y d e s o l u t i o n . T h e l u t h e r p r o c e s s is i d e n t i c a l w i t h t h e f~eces e s t i m a t i o n . H e r e t h e r e d u c t i o n figures are :~ 1. U r i n e t o r e d u c t i o n f l u i d , 8 0 to 120 = 1"5 × 2. F i l t r a t e t o s o l u t i o n , 2 0 t o 100 = 5 × 3. P e t r o l e t h e r s o l u t i o n t o " e n d f l u i d " 6 0 to R a n d t h e p e r c e n t a g e r e a d i n g is a g a i n m u l t i p l i e d to o b t a i n t h e u n i t s o f c o n c e n tration present. Again
iVy
× U
× 0"4 = r a g . o f u r o b i l i n , p r e s e n t i n 2 4 - h o u r s '
specimen
;
where V = volume of urine in c.cm., U = units of concentlation-present. IV,--CASE
HISTORIES.
C A S E I.
Male, 41. Railway Engineer. Born in Mauritius. Has been in India nineteen years, twelve years in Bombay. Personal History.--Several attacks of malaria, none severe, in last twelve years. Present illness began with diarrhoea. For four years prior to this had had two loose~ normal-coloured stools every morning between 6 and 8 o'clock. In May, 1925, ten to twelve motions per diem, pale and frothy liquid ; several in early morning, but some periods of exacerbation with diarrhoea all day. Tongue was sore at onset, with " aphth~e," but got better till November, 1925, when diarrhoea and tongue symptoms both became markedly severe. Flatulence and loss of energy combined to make him give up work. Treated in bed with milk and orange juice. Improved to January, 1926, when he was able to resume work. Arrived in England middle of March, 1926. Loss of weight, 38 lbs. (12 st. 6 lbs. to 9 st. 12 Ibs.). Complaint o1~ Admission.--Looseness of bowels, soreness of tongue, flatulence, loss of weight. Examination.--Well built, but obviously has lost weight. Skin, rather earthy tint ; mucous membranes, not anaemic ; tongue, fungiform papillae at tip are red and prominent, and are sore ; teeth and gums, healthy ; heart and htngs, nothing abnormal discovered ; pulse, regular, 80, equal ; distension of lower half of abdomen to slight degree, tympanitic to percussion ; liver, dulness slightly diminished above and below; spleen, not enlarged; sigmoidoscopy, mucous membrane lax and covered with dull mucus ; wall attenuated and rather pale ; no other abnormality detected in abdomen ; central nervous system, nothing abnormal detected ; Wassermann reaction, negative ; urine, 1016, acid ; no albumin ; no sugar ; mucus deposit only ; daily tests for urobilin and urobilinogen negative ; stools, pale, bulky, foul odour, sometimes frothy ; microscopically no ova nor protozoa, but numerous fat crystals. Blood.--lst April, 1926, red blood corpuscles, 4,300,000 ; h~emoglobin, 80 per cent. ; white blood corpuscles, 7,200 ; polymorphonuclears, 64 per cent. ; tymphocytes, 30 per cent. ; large mononuclears, 3 per cent. ; eoslnophiles, 3 per cent. ; no parasites seen. On l l t h April, red blood corpuscles, 4,600,000; hzemoglobin, 85 per cent. ; and on 20th April, red blood corpuscles, 5,000,000 ; hmmoglobin, 90 per cent. ; white blood corpuscles, 7,800.
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
9,95
Course.--Steady i m p r o v e m e n t on strict bed a n d diet. A t time of observation was o n dally diet as follows : milk or B e n g e r ' s food to 4 pints, juice of one orange, o n e egg, liver s o u p 8 ozs., sago p u d d i n g 6 ozs., r u s k s or toast (dry) 4 ozs. W e i g h t stationary, 62"7 kg. T h i s is a typical case of severe a l i m e n t a r y s p r u e w i t h o u t anaemia, or t h e p r i m a r y stage o f VAN
DER BURG. CASE II.
Male, 61. Retired Civil Servant. M a r r i e d . Born in E n g l a n d . I n Straits S e t t l e m e n t s f r o m 1888 to 1919. Personal History.--Very f r e q u e n t attacks of malaria u p to 1918 ; n o n e since. Indefinite diar~hoeic attacks since 1917 ; very m u c h worse in 1919, w h e n invalided h o m e ; stools n o t at that t i m e a b n o r m a l in colour, b u t very b u l k y ; repeated exacerbations every few m o n t h s till April. 1920, w h e n diarrhoea definitely white, copious, frothy, lasting all day b u t worse in t h e m o r n i n g . T o n g u e became very sore, w i t h excoriated p a t c h e s a n d a p h t h m . N o t very severe loss of weight at first, b u t later this b e c a m e m o r e a n d m o r e m a r k e d . D i a g n o s e d at this t i m e as s p r u e a n d treated with milk a n d d r u g s . I m p r o v e d in every way, b u t still h a d traces of disease in t h e f o r m of t r a n s i e n t attacks of diarrhoea. G r a d u a l loss of w e i g h t again in N o v e m b e r , 1925, a n d increased pallor. W a s on a very slightly modified diet. Pyrexial attack d i a g n o s e d as influenza (January, 1926), after w h i c h ( M a r c h , 1926), h e h a d r e t u r n of.diarrhoea ; pale, copious, s e m i - f o r m e d or f r o t h y stools, with attacks of n a u s e a a n d v o m i t i n g . T o n g u e sore b u t no aphth~e. Vertigo a n d altered m e n t a l i t y w i t h disorientation o f time a n d place. L o s s of w e i g h t extreme. Condition on Admission (24th M a r c h , 1926), was very grave. Examination.--Anmmia p r o f o u n d a n d emaciation e x t r e m e ; s e m i - c o m a t o s e . Tongue, papilhe enlarged a n d glazed ; heart, s o u n d s very faint, apex beat displaced to left b y p e r c u s s i o n ; radial pulses very weak ; very slight distension of a b d o m e n ; liver, d u l n e s s n o t d i m i n i s h e d ; spleen, n o t enlar.ged ; central nervous system, a p a r t f r o m cerebration, n o t h i n g a b n o r m a l detected ; Wassermann reactzon, negative ; weight, 52'75 kg. ; urine, 1012 acid, traces of a l b u m i n at first b u t later this disappeared ; n o s u g a r (for urobilin, see chart) ; stools, pale, bulky, s e m i - f o r m e d or f r o t h y ; fat crystals, b u t no ova or protozoa present. Blood.---25th M a r c h , 1926, erythrocytes, 1,100,000; leucocytes, 2 , 0 0 0 ; h~emoglobin, 30 p e r cent. ; p o l y m o r p h o n u c l e a r n e u t r o p h i l e s , 42 per cent. ; l y m p h o c y t e s , 56 p e r cent. ; large m o n o n u c l e a r s , 1 per cent. ; eosinophiles, 1 per cent. ; anisocytosis, poikilocytosis, a n d nucleated reds (normoblasts). O n 6th April, erythrocytes, 1,800,000; leucocytes, 2 , 4 0 0 ; h m m o g l o b i n , 30 per cent. O n 13th April, erythrocytes, 2,000,000 ; hzemoglobin, 40 per cent. ; on 20th April, erythrocytes, 2,430,000 ; h~emoglobin, 60 p e r cent. ; a n d on 27th April, erythrocytes, 3,760,000 ; h~emoglobin, 70 p e r cent. Course.--Treated in bed w i t h p e p t o n i s e d milk, glucose a n d B r a n d ' s essence of beef, a n d t r a n s f u s i o n s of blood (on 29th M a r c h a n d 9th April, 1 9 2 6 - - 5 0 0 c.cm. a n d ~00 c.cm. respectively). Slow b u t gradual i m p r o v e m e n t in general condition a n d blood picture. T h i s is a typical case of t h e s e c o n d stage of s p r u e (VAN DER BURO) in w h i c h t h e cardinal s y m p t o m s are p r e s e n t b u t t h e h~emolysis a n d blood d e s t r u c t i o n proceeded apace. CASE III. Male, 48. Civil Servant. M a r r i e d . B o r n in E n g l a n d . T w e n t y - n i n e years ago w e n t to C h i n a , J a p a n a n d Korea. T w e n t y - f i v e to t w e n t y - o n e years ago in E n g l a n d . F o r last t w e n t y - o n e years in Hong Kong. Personal History.--Malaria, 1907 to 1910 a n d in 1921 ; d y s e n t e r y (? variety) in 1910 a n d 1920. I n 1921, on s t e a m e r , gradual o n s e t o f diarrhoea, in m o r n i n g s , two or t h r e e loose stools b e c o m i n g white, frothy, b u l k y a n d offensive ; t o n g u e a n d m o u t h sore at this t i m e ; lost weight rapidly ; p r e s e n t weight, 60 kg., diarrhoea increased to seven stools daily b y 1925, w h e n ( N o v e m b e r ) blood a n d m u c u s a p p e a r e d for a s h o r t t i m e ; anaemia b e g a n to be severe ; h a d to give u p work a n d return to E n g l a n d . Condition on Admission (Sth April, 1 9 2 6 ) . - - E m a c i a t e d a n d e x t r e m e l y pale. Examination.--Shin, earthy t i n t a n d inelastic ; tongue, clean, b u t glazed a n d w i t h p r o m i n e n t " s h a v e n " f u n g i f o r m papillae, was n o t sore ; teeth, poor ; throat, clear ; lungs a n d heart, clear ; abdomen, wall t h i n a n d flaccid ; s u b c u t a n e o u s fat a b s e n t ; distension of lower half, m o r e pron o u n c e d before m o t i o n s passed ; liver, d i m i n i s h e d to h a n d ' s b r e a d t h in r i g h t m a m m a r y line ; splee~t, n o t enlarged ; sigmoid colon, t h i c k e n e d a n d palpable ; legs, s h o w old p i g m e n t e d scars of varicose ulcers ; central nervous system, clear ; urine, 1008, acid, faint trace a l b u m i n o n 9th April, 1926, n o n e since 16th April ; no sugar, a n d n o t h i n g pathological n o t e d in d e p o s i t ; stool, no ova nor protozoa seen ; m u c u s present, pale, greasy, offensive, f o r m e d a n d often constipated. T h e constipated stools were definitely b r o w n in colour. Blood.--9th April, 1926, red blood corpuscles, 2,000,000 ; h~emoglobin, 60 p e r cent. ; w h i t e blood corpuscles, 5 , 2 0 0 ; colour i n d e x , 1"5; p o l y m o r p h o n u c l e a r n e u t r o p h i l e s , 72 p e r cent.
296
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
lymphocytes, 26 per cent. ;. large mononuclears, 2 per cent. ; poikilocytosis, anisocytosis, and one normoblast seen ; no paramtes present. O n 27th April, red blood corpuscles, 3,200,000 ; heemoglobin, 70 per cent. ; eolour index, 1"1 ; Wassermann reaction, negative. Course.--Slow i m p r o v e m e n t in general condition, with rapid i m p r o v e m e n t in appearance of stool. T r e a t e d in bed with diet. D u r i n g period of observation diet was 6 pints of milk, 2 ozs. rusks, 4 ozs. sago pudding, and one egg daily. T h i s is a second case of long-continued sprue w h e r e the blood destruction had become profound ; in some respects like Case I I , b u t the process had been m o r e gradual and less severe. T h e blood picture resembled Addisonian anmmia, b u t clinically the case was typically s p r u e without marked alimentary s y m p t o m s .
V.--TABULATED
RESULTS.
CASE I--F~ECES,
Weight of Stool (in grammes).
Days.
Total Urobilin and Urobilinogen (in milligrammes).
1st
...
2nd 3rd 4th 5th 6th 7th
... ... ... ... ... ...
219 574 222 182 235 215 275
9o 138 196 98 288 193 259
Total
...
1,922
1,262
Urobilinogen (in milligrammes of Urobilin).
N o t estimated. J 81 205 142 166
I
Average pe~ day
274"5
594
180
148'5
Percentage present as Urobilinogen = 71. Quantity of Urobilin per 100 g. fmces = 67"5 rag. Estimations of urine not made, as qualitative tests uniformly negative. CASE II--F~CE$.
Days.
Weight of Stool (in grammes),
Total Urobilin and Urobilinogen (in milhgrammes).
Urobilinogen (in milllgrammes of Urobilin).
115 245
185 537
128 518
Xst
...
2nd 3rd 4th 5th 6th 7th
... ... ... ... ... ...
614 17
1,326 9
1,229 7
611
745
687
Total
...
1,602
2,802
2,569
229
400
367
Average per day
Percentage present as Urobilinogen = 91"75. Quantity of Urobilln per 100 g. fieces ~ 174"7 rag.
SOME OBSERVATIONS ON U R O B I L I N
OUTPUT
IN SPRUE
297
CASE. I I - - U R I N E .
Day s .
Q u a n t i t y in cubic centimetres.
Urobilin ix milligrammes.
Schlesinger's Test.
Ehrlich's Test.
++ + ++ ++ ÷ + +
1st 2nd 3rd 4 th 5 th 6th 7th
... ... ... ... ... ... ...
1,930 1,400 2,125 2,555 1,870 1,540 2,330
9 4 8 15 2
÷ + + + + weak +
Total
...
13,750
41
--
__
1,964
6
--
--
A v e r a g e p e r day
2 1
T o t a l d a i l y e x c r e t i o n of U r o b i l i n = 406 rag.
CASE III--FfiECES.
W e i g h t of Stool in g r a m m e s ) .
Days.
Total Urobilin and U r o b i l i n o g e n (in r n i l l i g r a m m e s of U r o b i l i n ) ....
I
1st
.,.
2nd 3rd 4th 5th 6th
... ... ... ... ...
347 165 220 182 140 184
TotaJ
...
1,238
:
U r o b i l i n o g e n (in m i l l i g r a m m e s of Urobilin).
454 158 306 227 254 306
286 154 174 178 195 179
1,705
1,166
284
194
I A v e r a g e p e r day
206'3
P e r c e n t a g e p r e s e n t as U r o b i l i n o g e n = 68. Q u a n t i t y of U r o b i l i n per 100 g. f~eces = 137"7 m g .
298
SOME OBSERVATIONS ON UROBII.IN OUTPUT IN SPRUE
CASE III--1)IIINE.
Q u a n t i t y l n cubic centimetres,
Days.
U r o b i l i n in milligrammes.
1st
...
2nd 3rd 4th 5th 6th
... ... ... ... ...
2,300 2,500 1,720 2,120 2,775 1,920
2"4 1'6 0'8 2'0 2"5 2"8
Total
...
13,335
12"1
Averag e p er day
2,222'5
Ehrlich's Test.
÷ ÷
Schlesinger's Test.
÷
weak
+ ÷ +
÷ ÷
2"0
T o t a l daily excretion of U r o b i l i n = 286 mg.
DISCUSSION.
In the past, many statements have been made with regard to the cause of the colour of sprue stools. Most of these explanations have taken for granted that the absence of the normal colour had to be accounted for by showing the bile-pigment products to be present in an altered form. VAN DER SCHEER (1905) states that except for traces of unreduced bilirubin, all the bile-pigment is present as leuko-urobilin (NENCKI). DOLD (1924) repeated this statement, held a substance responsible by the same name and defined it as a colourless chromogen of urobilin. In English text-books on Tropical Medicine the substance is mentioned, with occasional changes of name to leucobilin in which form it is even harder to recognise. Now, although not uncommon in German text-books of physiology as a clinical term, none of these names figure in modern English or American physiological chemistry works, and their use leads some students to consider that in sprue one has to deal wlth a new and peculiar product of hepatic (or enteric) activity. This opinion, indeed, is shared by some authorities on the disease, who invariably quote SCHMIDT'S corrosive sublimate test as a proof of the presence of bile-pigment products. M A N S O N - B A H R brought forward interesting features in his work in Ceylon, and showed that fresh sprue stools did not give a positive Schmidt's reaction until some time had elapsed. In a footnote, he quotes STRASBURGERas maintaining the identity of NENCKI'S leucourobilin and the substance ordinarily known as urobilinogen.
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
9@9
On referring to the figures obtained in the three cases studied, it will be seen that :-1. In Case I, without severe anmmia, the quantity of total urobilin present (t80 mg.) is only slightly higher than the average normal (140 mg.) (LICHTENSTEIN, 1924 ; CHARNAS, 1909) ; but of this total 71 per cent. is a colourless compound, and the remaining 29 per cent. is distributed throughout a stool of average daily weight 274"5 g., which is double the normal. Hence, the dilution of the actual pigment is approximately six times. 2. In Case II, whilst the average daily weight of f~eces is only one and a half times the normal, the quantity of total urobilin is much increased. The more interesting feature is that the percentage of pigment present as the chromogen is 91"75 ; hence, the actual dilution of pigment is eighteen times. LICHTENSTEIN, from his series of observations, states that the quantity of urobilin excreted in 100 g. of f~eces seldom exceeds 120 rag. ; which figure compares with the 174"7 mg. in this case, in spite of the fact that the total quantity of f~eces per diem is simultaneously increased. 3. In Case III we have an intermediate stage, in which more of the nrobilinogen has passed over into the oxidised state than in Cases I and II. The proportion of urobilinogen, however, is high (68 per cent.). The dilution is much less--about four times--thus agreeing with the appearance of the stool. Here, then, we have figures showing that the ratio of urobilinogen to urobilin is very high in sprue, and that the actual amount is much increased in those cases with anaemia. As the form in which the chromogen is present allows it to be measured by a urobilinogen reaction, the suggestion is made that, in future, leucobilin and similar synonyms be avoided and the more familiar term urobilinogen used. It is not proposed to assert that this is the complete explanation of the colour of sprue stools, since the well-known experiment of HERMANN must be kept in mind. This matter of the Hermann isolated loop of intestine and its secreted f~eces is one of great difficulty ; STARLINGstates "After some weeks an isolated loop (of intestine with full blood supply) will be found to contain a semi-solid material similar to fa~ces in appearance, consistence and chemical constitution," on which experiment is based the statenlent in many physiology books, that the normal colour of f~eces has no relation to the presence of bilepigments and their products. SCHMIDT and STRASBURGER,however, quote EHRENTHAL thus : Hermannsche Ringkot ungefarbt, grau aussieht. In this uncertain position it is as well to remember that, in sprue, we h~,ve a proof of the effect of bile-pigment products in producing a colour approaching normal in the stool, for, as has long been known to clinicians, the greyish stool of sprue gradually assumes a brownish tint when exposed to light and air, a change due to the oxidation of urobitinogen to urobilin.
800
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
VI.--FURTHER EXPERIMENTS. In order to confirm these high ratios of urobilinogen to urobilin, further experiments were performed ; two on fresh specimens of sprue stool (within half an hour of their being passed) and two, on consecutive days, on a normal patient on whom a plastic surgical operation had been p e r f o r m e d . SPRUE CASE A, Chronic type ; firm, pasty stool, Wasting and previous history of sore tongue ; anemia ; red blood corpuscles, 2,800,000 ; hmmoglobin, 65 per cent. ; weight of stool, 128"28 g. ; urobilinogen, 215 rag. ; total urobilin and urobilinogen, 235 mg. ; urobilin, 20 rag. Urine gave negative Schlesinger and very weak Ehrlich. In this case, 91"5 per cent. of the bile-pigment product in a fresh sprue stool was present as urobilinogen. SPRUE CASEB. One and a half years' duration ; fluid, slightly pigmented, frothy stools ; n o t m u c h wasting ;
tongue symptoms very slight ; liver diminished in size ; red blood corpuscles, 4,700,000 ; hmmoglobin, 85 per cent. ; weight of stool, 101'98 g. ; urobilinogen, 78 rag. ; total urobilin and urobilinogen, 104 mg. In this case, 75 per cent. of the product was present as the colourless chromogen. SURGICALCASE. Stool normal in colour and appearance. On milk, fish, bread, etc. First Day.--Weight of stool, 84"25 g. ; urobilinogen, 84 rag. ; total urobilin and urobilinogen, 165 mg. Second Day.--Weight of stool, 58 g. ; urobilinogen, 27 rag. ; total urobilin and urobilinogen, 98 mg. Daily Average.--Weight of stool, 71 g. ; urobilinogen, 55"5 rag. ; total urobilin and urobilinogen, 131"5 rag. In this case, 42'2 per cent. of the product was present as urobilinogen.
Unfortunately. there are no other figures available of this percentage in fresh, normal stools. V I I . - - T H E RELATION OF UROBILIN OUTPUT TO BLOOD BREAKDOWN. T h e total urobilin o u t p u t was first employed by EPPINGER (1920) to obtain a measure of the rapidity of blood destruction, but he made use of a factor which he stated to represent the normal a m o u n t of bilirubin secreted per diem. This factor he had obtained by carefully weighing the material obtained from two patients with complete bile fistulm. T h e theoretical figure thus obtained did not, however, coincide with those obtained in practice. T o obviate this, LICHTENSTEIN (1924) has introduced an " index of blood destruction which he defines as " the rapidity with which a patient destroys his blood compared with that of a normal person." T o obtain this index, LICHTENSTEIN argues thus : " T h e weight of the patient in kg. is obtained easily ; m o d e r n figures agree that approximately one-twentieth of this weight is blood. Of the blood, 14 per cent. by weight normally is h~emoglobin and 4"47 per cent. of the h~emoglobin is h~ematin. T h u s , the total quantity of h~ematin in any person can be calculated if their weight be known, and can be adjusted more accurately if the percentage of hmmoglobin in the blood be estimated in a reliable manner. At the same time, the average daily a m o u n t of urobilin excreted can be measured, and from this figure the equivalent amount of h~ematin per day calculated. "
SOME OBSERVATIONS ON UROBILIN
OUTPUT
IN SPRUE
301
T h u s , i n a m a n of 70 kg. w e i g h t , the total h~ematin i n t h e b l o o d c a n be c a l c u l a t e d as 21"9 g. I f t h e u r o b i l i n e x c r e t e d p e r d i e m be t h e n o r m a l 150 m g . (LICHTENSTEIN), t h e n this is calculated to b e d e r i v e d f r o m 153 m g . of h~ematin. T h u s all the h~ematin c o u l d b e d e s t r o y e d i n a b o u t 140 days. A p p l y i n g this to t h e f o r e g o i n g cases : CASE I. Body weight, 62"7 kg. ; hzemoglobin, 85 per cent. ; weight of blood, 3,135 g. ; weight of h~emoglobin, 0"85 × 0"14: × 3,135, or 373 g. ; weight of h~ematin, 0"044:7 × 373, or 16"673 g. ; average daily output of urobilin, 183 rag. ; equivalent to ~0 × 183 or 186"7 rag. of hmmatin. Therefore, the time taken is ~ . , ~ X 1 or 89 days. The corresponding time for a normal man of the same weight, with 100 per cent. of h~emoglobin, and excreting 150 mg. of urobilin per diem, is 128 days. Therefore, the index is l~s or 1"4. That is, he is using his blood 1"4 times as rapidly as a normal man. CASE I I .
Body weight, 52"75 kg. ; hmmoglobin, 55 per cent. ; weight of blood, 2,637"5 g. ; weight of hmmoglobin, 0"55 × 0"14 × 2,637"5, or 203"088 g. ; weight of hmmatin, 9"078 g. ; average daily output of urobilin, 406 rag. ; equivalent to ~xo x 406 or 414 mg. of hmmatin. Therefore, the time taken is 0~7~ or 22 days, approximately. The corresponding time for a normal man of the same weight, with 100 per cent. of hmmoglobin and excreting 150 rag. of urobilin per diem, is 108 days. Therefore the index is ½0s or 4"9. CASE III.
Body weight, 60 kg. ; h~emoglobin, 70 per cent. ; weight of blood, 3,000 g. ; weight of h~emoglobin, 0"7 × 0'14 × 3,000, or 294 g. ; weight of hmmatin, 294 × 0'0447 or 13"142 g. ; average daily output of urobilin, 286 rag. -, equivalent to ~'~l × 286 or 291"7 mg. of hmmatin. Therefore, the time taken is a~#{ or 45 days, approximately. The corresponding time for a normal man is I22'7 days. Therefore, the index is -~.ea~ or 2"7. VIII.--SUMMARY. 1. E s t i m a t i o n s of b i l e - p i g m e n t p r o d u c t s b y the m e t h o d of T~RWEN, have b e e n carried o u t o n t h r e e cases of s p r u e , i n each case e s t i m a t i o n s b e i n g c o n t i n u e d daily for o n e week. 2. I n two cases w i t h severe anaemia t h e average daily o u t p u t o f " u r o b i l i n '~ ( u r i n e a n d f~eces) was m u c h a b o v e n o r m a l . I n t h e case w i t h o u t m a r k e d anaemia, the o u t p u t was w i t h i n t h e l i m i t s of n o r m a l . 3. T h e p e r c e n t a g e of t h e total p r o d u c t s p r e s e n t as u r o b i l i n o g e n - - t h e colourless p r e c u r s o r of u r o b i l i n - - v a r i e d f r o m 68 to 91"75, a n d these figures were c o n f i r m e d i n fresh stools f r o m two f u r t h e r s p r u e p a t i e n t s . 4. T h e p e r c e n t a g e of t h e total of t h e bile p i g m e n t p r o d u c t s p r e s e n t as u r o b i l i n o g e n i n a c o n t r o l surgical case was 42"2. 5. T h e i n d e x of b l o o d d e s t r u c t i o n (LICHTENSTEIN) has b e e n w o r k e d o u t o n t h e t h r e e m a i n cases a n d the results s h o w t h a t t h e y b r e a k d o w n t h e i r b l o o d 1"4, 4"9, a n d 2"7 t i m e s as r a p i d l y as the n o r m a l case is c a l c u l a t e d to do. 6. T h e s u g g e s t i o n is m a d e t h a t the t e r m s l e u c o - u r o b i l i n a n d l e u c o b i l i n , as at p r e s e n t u s e d , b e r e p l a c e d b y the t e r m u r o b i l i n o g e n , as the s u b s t a n c e p r e s e n t i n t h e f~eces i n s p r u e gives all the characteristic r e a c t i o n s of this g r o u p .
809.
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
In conclusion I wish to thank Drs. G. C. L o w and P. H. MANSON-BAHR for access to their cases in the Hospital for Tropical Diseases, W.C.1, and to the Resident Staff at the Hospital for their whole-hearted co-operation. To Dr. H. B. NEWHAM, C.M.G., I am indebted for permission to work in his laboratory and for his ready help and guidance at all stages of the work.
REFERENCES. CI-IARNAS,D. (1909). Ueber die Darstellung, das Verhatten und die quantitative Bestimmung des reinen Urobilins und des Uribilinogens. Biochern. Ztschr. Berl. xx, 401. BEAUMONT, G. E. and DODDS, E. C. (1924). Recent Advances in Medicine. DOLD, H. (1924). Die Spru (Tropische Aphthen). Handbuch der Tropenkranhheiten, edited by CARL ~V~ENSE. (3rd Edition). ii, 402. Leipzig. EPPINGER, ]7I. (1920). Die Hepatolienalen Erkrankungen. HOPPE-SEYLER,G. (1891). Ueber die Ausscheidung des Urobilins in Krankheiten. Arch. Path. Anat. und Phys. cxxiv, 30. JAFFE, M. (1869). Zur Lehre yon den Eigensehaften und der Abstammung der Harnpigmente. Arch. Path. Anat. und Phys. xlvii, 405. LICHTENSTEIN, A. (1924). Quantitative Urobilin bepaling en Bloedafbraah. Amsterdam. MALY, l~. (1871). Kunstliche Umwandlung yon Bilirubin in Harnfarbstoff. Zentralblatt. f . d. Med. Wissen. ix, 849. MANSON-BAHR, P. H. (1915). A Report on Researches on Sprue in Ceylon. 1912-1914. SA1LLET. (1897). De l'Urobiline dans les Urines Normales. Revue de Med. xvii, 109. STARLING,E. (1926). Principles of Human Physiology. 4th Edition, 630. London. SCI-IralDT, A. and STRASBURGER,G. (1910). Die Fa~es. 26 and 257. Berlin. TERWEN, J. L. (1924). De Quantitative Bepaling van Urobiline en Urobilinogen in Urine en F~eces. Amsterdam. VAN DER SCribER, A. (1905). Aphthae Tropicae. Handbuch der Tropenkranhheiten, edited by CARL MENSE. (lst Edition). ii, 1. Leipzig. VANLAIR and MASlUS. (1871). Ueber Einen Neuen Abkommling des Gallenfarbstoffs im Darminhalt. Zentralblatt f . d. Med. Wissen. ix, 369. VAN D•R BURT, C. L. (188l). Indische Spruw Batavia, (1880). Geneesk. T~jdschr. f . Ned. Indue. X~ l .
SOME OBSERVATIONS ON U R O B I L I N O U T P U T IN SPRUE. BY R. M. MORRIS, M.D. (Lond.), D.T.M. & H. ~
Salisbury, Southern Rhodesia.
I.--INTRODUCTION.
Our knowledge of urobilin dates from 1868, when JAFFE (1869) first discovered its presence in some urines. MALY (1871) obtained a product-hydrobilirubin--by a direct reduction of the bilirubin in bile, and was able to demonstrate its identity with JAFFE'S urobilin and the stercobilin, which VANLAIR and !V[ASIUS (1871) had procured from f~eces. HOPPE-SEYLER (1891) was one of the first to emphasize that in urobilin one had to deal with the normal, final, product of bile-pigments, which, in turn, were derived from the blood-pigment, haemoglobin. This point was utilized by EPPINgER (1920)', who sought in this way to obtain a quantitative estimate of blood destruction. In sprue, the interest in this substance is two-fold. Firstly, the peculiar and characteristic colour of the freshly-voided faeces in this disease excites interest in any matter which concerns stool-pigments, and secondly, the anaemia accompanying the severer cases is worthy of deep and continued study. Hitherto, whilst the qualitative tests for urobilin in urine or faeces have been a simple matter, the quantitative ones have involved laborious processes, with special apparatus ; but recently TERWEN (1924) introduced a simple colorimetric method, which insures reliable results and is easily applicable in an ordinarily-equipped laboratory. It is with the aid of this method that the data in this paper have becn obtained. II.--PROPERTIES OF UROBILIN AND UROBILINOGEN.
Before proceeding further, it may not be out of place to recall some of the properties of these substances. JAFFE described how solutions of zinc salts of urobilin, in alcohol or chloroform, present a remarkable fluorescence, which appears pink by transmitted and green by reflected light ; on which property is based the usual Schlesinger -e Thesis approved for the Degree of Doctor of Medicine in the University of London. From the Hospital for Tropical Diseases, I.ondon, and the Department of Pathology, London School of Hygiene and Tropical Medicine.
SOME
OBSERVATIONS
ON
UROBILIN
OUTPUT
IN
SPRUE
291
test for urobilinuria. The other main test is the absorption band in the greenblue area of the spectrum--the exact position varying slightly according to the reaction and nature of the solvent. The zinc salts also have a characteristic band in the same area. Urobilin itself is a labile substance, which hitherto has been prepared only as an amorphous, reddish-brown powder of uncertain purity, and it has been shown that the above criteria are group reactions, not specific; hence, chemically, urobilin may be of several varieties; clinically, however, this is of no moment. Another point noted by JAFEEwas that, in the urines which gave the fluorescent test, the dark colour did not appear till they had been standing for some time ; he, therefore, postulated a colourless chromogen. Later, this substance was isolated from urine and named urobilinogen. It has been prepared in crystalline form and shown to be a reduction product of urobilin, into which it returns under influence of light and air (SAILLET,1897). The relationship may be shown graphically thus :-Bilirubin-----~- Urobilin ~ Urobilinogen. The main test for urobilinogen is the Ehrlich's aldehyde reaction, where a deep red colour is imparted to the urine by the addition of a few drops of 3 per cent. paradimethylamino-benzaldehyde in 50 per cent. hydrochloric acid, which red colour presents an absorption band in the green. A second test is a diazo-reaction, where Ehrlich's diazo-reagents produce an orange colour in the urine, changing to a citron-yellow on adding ammonia. These reactions again are " group reactions," but for clinical purposes suffice. III.--METHODS
EMPLOYED.
URINE.--All the tests were carried out on 24-hours' specimens of urine collected in a black bottle. As a routine, Ehrlich's aldehyde reaction for urobilinogen and Schlesinger's test for urobilin were performed directly the 24hours' specimen was received. If positive, the total quantity was measured and a portion set aside for quantitative examination, in which no attempt was made to differentiate the proportions of urobilin and urobilinogen. F~EcES.--These estimations were also carried out on 24-hours' specimens, collected from noon to noon and kept in an opaque jar, with a tightly-fitting lid, and in a dark corner. The total quantity was then weighed and the whole mass rendered as homogenous as possible with a pestle. Two portions of 5 g. each were then weighed out as rapidly as possible and the estimations carried out on them. One portion was used for the estimation of the quantity of urobilinogen and the other for the total urobilin plus urobilinogen present. Each patient's excreta were examined daily for 7 days. TE~WEN'S method is a colorimetrie one in which, first, all the urobilin present is reduced to urobilinogen, the latter extracted in a suitable solvent, and the aldehyde reaction performed with this solution. The intensity of the colour developed is then
29~
SOME OBSERVATIONS ON UROBILIN OUTPUT I N SPRUE
compared with that of a standard, alkaline, phenolphthalein solution in a colorimeter, and this result expressed in units of concentration. Gravimetrically, TERWEN has shown one unit of concentration to be equal to 0"4 mg. urobilin per 100 c.cm. urine or 100 g. f~eces. If the reduction step in the process be omitted and a selective solvent, e.g'., petroleum ether, which extracts urobilinogen but not urobilin, be used, a quantitative estimate of the amount of the chromogen alone can be obtained. DETAILS OF PROCEDURE.
THE F ~ c E s . - - T h e 5 g. of f~eces are made up into a homogeneous emulsion with 50 c.cm. of distilled water and 50 c.cm. of a freshly prepared 16 per cent. Mohr's salt (FeSO4, (NH4)2SO4, 6H20 ) solution added. Then, in a fine stream from a pipette, 50 c.cm. of 12 per cent. solution of caustic soda are allowed to run in, the beaker being constantly agitated. This results in a greenish, turbid liquid, which is forthwith poured into a flask so that a glass stopper can be inserted without the presence of an air bubble. The whole is then put away in a dark cupboard for at least eighteen hours, during which time the urobilin should be reduced to urobilinogen. That this has duly taken place must be controlled by the spectroscope, when the urobilin band will be absent if reduction has been complete. The fluid is then filtered through a pure, dry filter into a brown flask. The rest of the process must then be carried out without interruption, as the urobilinogen tends to pass into urobilin by the oxidising action of air and light. 5 c.cm of the filtrate are then accurately measured and placed in a clean, dry, separating funnel fitted with a stopper, in which it must be rendered acid by the addition of 2"5 c.cm. of a 20 per cent. tartaric acid solution. (This may be controlled with litmus paper). Into the separating funnel is then measured 40 c.cm. of petroleum ether (s.g., 0"65 ; boiling point 40 ° to 60 ° C.) and the whole shaken up about seventy times. On separation of the layers, the lower may be allowed to run off, as the urobilinogen will have passed into the petrolether. From this latter, 20 c.cm. are measured off into a second, clean, dry, separating funnel, and 20 c.cm. of ether added. (This ether must be p u r e - commercially pure ether washed with caustic soda and distilled water and then distilled.) To the mixture are added 5 c.cm. of a saturated solution of paradimethylamino-benzaldehyde (MERci() in purified ether. Rapidly after this 0"5 c.cm. of 38 per cent. hydrochloric acid is added and the whole vigorously shaken for one and a quarter minutes, when a few c.cm. of water--recently distilled--are blown in from a wash bottle. The shaking is continued for another fifteen seconds, and then 3 c.cm. of a saturated solution of sodium acetate are added. This causes a milky appearance, which disappears on further vigorous shaking. The purpose of the acetate is to remove the colour reactions due to indol and skatol bodies, which, having unsaturated
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
298
pyrrol-rings, also give red or reddish-blue colours with Ehrlich's reagent (e.g., the cholera-red reaction). The now clear, reddish-blue fluid in the lower layer is allowed carefully to run out into a measuring flask. A further portion of urobilinogen can be made to react by repeating the process from the addition of the hydrochloric acid, and the resulting fluid is added to that previously obtained. The quantity is then measured and the fluid is used in a colorimeter. TERWEN uses an Authenrieth colorimeter, for which a curve is plotted, but nearly as accurate results may be obtained by using a Dubosc instrument and giving the result as a percentage of one unit of concentration, one unit being defined as the concentration which gives a colour, in an " end-fluid " of 10 c.cm., exactly matching the colour of the standard. This standard, which must be freshly prepared each day, consists of 5 c.cm. of a 19 per cent. anhydrous sodium carbonate solution (saturated), 1 e.cm. of a 0"05 per cent. solution of phenolphthalein in alcohol, and 94 c.cm. of freshly distilled water. Further, in calculating the result, the progressive dilutions must be taken into account, vi~. :-1. From f~eces to reduction fluid 5 to 150 -- 30 × 2. From filtrate to petrol-ether solution, 5 to 40 = 8 × 3. From petrol-ether solution to " end fluid." 20 to R. and the percentage reading must be multiplied by these. This gives the result in units of concentration, from which the weight in milligrammes of urobilin in the stools can be obtained thus :-W × U × 0"4 = mg. of urobilin per diem ; xv~ w h e r e W = w e i g h t of 24 h o u r s ' f~eces in g r a m m e s , U -- u n i t s of c o n c e n t r a t i o n pre s e nt .
The reaction for the quantity of urobilinogen present is performed in exactly the same way, except that the emulsion of the 5 g. of f~eces is made up to 150 c.cm. with distilled water and the reduction process is omitted, the diluted f~eces being immediately filtered. The result is expressed as urobilinogen in terms of mg. of urobilin. THE URINE.--The Qualitative Tests.
Urobilin--Schlesinger's Test.--20 c.cm. of urine are acidulated with acetic acid and the urobilin extracted by gently inverting with 5 c.cm. of amylic alcohol. This layer is then pipetted into a second clean, dry, test-tube and a few drops of 10 per cent. alcoholic solution of zinc chloride added. If positive, a green fluorescence develops and an absorption band in the green-blue may be seen with a hand splectroscope. This indicates an excess of urobilin. Urobilinogen--Ehrlich's Aldehyde Reaction.--To 5 c.cm. of urine 2 drops of a 3 per cent. solution of paradimethylamino-benzaldehyde in 50 per cent. hydrochloric acid are added. A deep red colour gradually makes its appearance,
"29~
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
more readily if warmed slightly. In this also can an absorption I t is p o s i t i v e w i t h a n e x c e s s o f u r o b i l i n o g e n . Quantitative
band
be s e e n .
Test.
N o a t t e m p t w a s m a d e to d i f f e r e n t i a t e t h e t w o s u b s t a n c e s . The process is v e r y s i m i l a r to t h a t o f t h e f~eces, b u t 8 0 c . c m . o f u r i n e , f r o m a w e l l - s h a k e n 2 4 - h o t l r s ' s p e c i m e n , a r e u s e d , a n d t h e r e d u c t i o n c a r r i e d o u t w i t h 20 c . c m . of t h e M o h r ' s salt s o l u t i o n a n d 20 c . c m . c a u s t i c s o d a . A g a i n it m u s t b e l e f t in t h e d a r k , w i t h all air e x c l u d e d , f o r at l e a s t e i g h t e e n h o u r s . From the filtrate, 20 c.cm. are taken and acidulated with 5 c.cm. tartaric acid and the extraction performed with 80 c.cm. of petroleum ether, of which 60 c . c m . a r e u s e d i n t h e s e c o n d s e p a r a t i n g f u n n e l . T h e q u a n t i t y o f e t h e r a d d e d is 60 c . c m . a n d 5 c . c m . o f E h r l i c h ' s a l d e h y d e s o l u t i o n . T h e l u t h e r p r o c e s s is i d e n t i c a l w i t h t h e f~eces e s t i m a t i o n . H e r e t h e r e d u c t i o n figures are :~ 1. U r i n e t o r e d u c t i o n f l u i d , 8 0 to 120 = 1"5 × 2. F i l t r a t e t o s o l u t i o n , 2 0 t o 100 = 5 × 3. P e t r o l e t h e r s o l u t i o n t o " e n d f l u i d " 6 0 to R a n d t h e p e r c e n t a g e r e a d i n g is a g a i n m u l t i p l i e d to o b t a i n t h e u n i t s o f c o n c e n tration present. Again
iVy
× U
× 0"4 = r a g . o f u r o b i l i n , p r e s e n t i n 2 4 - h o u r s '
specimen
;
where V = volume of urine in c.cm., U = units of concentlation-present. IV,--CASE
HISTORIES.
C A S E I.
Male, 41. Railway Engineer. Born in Mauritius. Has been in India nineteen years, twelve years in Bombay. Personal History.--Several attacks of malaria, none severe, in last twelve years. Present illness began with diarrhoea. For four years prior to this had had two loose~ normal-coloured stools every morning between 6 and 8 o'clock. In May, 1925, ten to twelve motions per diem, pale and frothy liquid ; several in early morning, but some periods of exacerbation with diarrhoea all day. Tongue was sore at onset, with " aphth~e," but got better till November, 1925, when diarrhoea and tongue symptoms both became markedly severe. Flatulence and loss of energy combined to make him give up work. Treated in bed with milk and orange juice. Improved to January, 1926, when he was able to resume work. Arrived in England middle of March, 1926. Loss of weight, 38 lbs. (12 st. 6 lbs. to 9 st. 12 Ibs.). Complaint o1~ Admission.--Looseness of bowels, soreness of tongue, flatulence, loss of weight. Examination.--Well built, but obviously has lost weight. Skin, rather earthy tint ; mucous membranes, not anaemic ; tongue, fungiform papillae at tip are red and prominent, and are sore ; teeth and gums, healthy ; heart and htngs, nothing abnormal discovered ; pulse, regular, 80, equal ; distension of lower half of abdomen to slight degree, tympanitic to percussion ; liver, dulness slightly diminished above and below; spleen, not enlarged; sigmoidoscopy, mucous membrane lax and covered with dull mucus ; wall attenuated and rather pale ; no other abnormality detected in abdomen ; central nervous system, nothing abnormal detected ; Wassermann reaction, negative ; urine, 1016, acid ; no albumin ; no sugar ; mucus deposit only ; daily tests for urobilin and urobilinogen negative ; stools, pale, bulky, foul odour, sometimes frothy ; microscopically no ova nor protozoa, but numerous fat crystals. Blood.--lst April, 1926, red blood corpuscles, 4,300,000 ; h~emoglobin, 80 per cent. ; white blood corpuscles, 7,200 ; polymorphonuclears, 64 per cent. ; tymphocytes, 30 per cent. ; large mononuclears, 3 per cent. ; eoslnophiles, 3 per cent. ; no parasites seen. On l l t h April, red blood corpuscles, 4,600,000; hzemoglobin, 85 per cent. ; and on 20th April, red blood corpuscles, 5,000,000 ; hmmoglobin, 90 per cent. ; white blood corpuscles, 7,800.
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
9,95
Course.--Steady i m p r o v e m e n t on strict bed a n d diet. A t time of observation was o n dally diet as follows : milk or B e n g e r ' s food to 4 pints, juice of one orange, o n e egg, liver s o u p 8 ozs., sago p u d d i n g 6 ozs., r u s k s or toast (dry) 4 ozs. W e i g h t stationary, 62"7 kg. T h i s is a typical case of severe a l i m e n t a r y s p r u e w i t h o u t anaemia, or t h e p r i m a r y stage o f VAN
DER BURG. CASE II.
Male, 61. Retired Civil Servant. M a r r i e d . Born in E n g l a n d . I n Straits S e t t l e m e n t s f r o m 1888 to 1919. Personal History.--Very f r e q u e n t attacks of malaria u p to 1918 ; n o n e since. Indefinite diar~hoeic attacks since 1917 ; very m u c h worse in 1919, w h e n invalided h o m e ; stools n o t at that t i m e a b n o r m a l in colour, b u t very b u l k y ; repeated exacerbations every few m o n t h s till April. 1920, w h e n diarrhoea definitely white, copious, frothy, lasting all day b u t worse in t h e m o r n i n g . T o n g u e became very sore, w i t h excoriated p a t c h e s a n d a p h t h m . N o t very severe loss of weight at first, b u t later this b e c a m e m o r e a n d m o r e m a r k e d . D i a g n o s e d at this t i m e as s p r u e a n d treated with milk a n d d r u g s . I m p r o v e d in every way, b u t still h a d traces of disease in t h e f o r m of t r a n s i e n t attacks of diarrhoea. G r a d u a l loss of w e i g h t again in N o v e m b e r , 1925, a n d increased pallor. W a s on a very slightly modified diet. Pyrexial attack d i a g n o s e d as influenza (January, 1926), after w h i c h ( M a r c h , 1926), h e h a d r e t u r n of.diarrhoea ; pale, copious, s e m i - f o r m e d or f r o t h y stools, with attacks of n a u s e a a n d v o m i t i n g . T o n g u e sore b u t no aphth~e. Vertigo a n d altered m e n t a l i t y w i t h disorientation o f time a n d place. L o s s of w e i g h t extreme. Condition on Admission (24th M a r c h , 1926), was very grave. Examination.--Anmmia p r o f o u n d a n d emaciation e x t r e m e ; s e m i - c o m a t o s e . Tongue, papilhe enlarged a n d glazed ; heart, s o u n d s very faint, apex beat displaced to left b y p e r c u s s i o n ; radial pulses very weak ; very slight distension of a b d o m e n ; liver, d u l n e s s n o t d i m i n i s h e d ; spleen, n o t enlar.ged ; central nervous system, a p a r t f r o m cerebration, n o t h i n g a b n o r m a l detected ; Wassermann reactzon, negative ; weight, 52'75 kg. ; urine, 1012 acid, traces of a l b u m i n at first b u t later this disappeared ; n o s u g a r (for urobilin, see chart) ; stools, pale, bulky, s e m i - f o r m e d or f r o t h y ; fat crystals, b u t no ova or protozoa present. Blood.---25th M a r c h , 1926, erythrocytes, 1,100,000; leucocytes, 2 , 0 0 0 ; h~emoglobin, 30 p e r cent. ; p o l y m o r p h o n u c l e a r n e u t r o p h i l e s , 42 per cent. ; l y m p h o c y t e s , 56 p e r cent. ; large m o n o n u c l e a r s , 1 per cent. ; eosinophiles, 1 per cent. ; anisocytosis, poikilocytosis, a n d nucleated reds (normoblasts). O n 6th April, erythrocytes, 1,800,000; leucocytes, 2 , 4 0 0 ; h m m o g l o b i n , 30 per cent. O n 13th April, erythrocytes, 2,000,000 ; hzemoglobin, 40 per cent. ; on 20th April, erythrocytes, 2,430,000 ; h~emoglobin, 60 p e r cent. ; a n d on 27th April, erythrocytes, 3,760,000 ; h~emoglobin, 70 p e r cent. Course.--Treated in bed w i t h p e p t o n i s e d milk, glucose a n d B r a n d ' s essence of beef, a n d t r a n s f u s i o n s of blood (on 29th M a r c h a n d 9th April, 1 9 2 6 - - 5 0 0 c.cm. a n d ~00 c.cm. respectively). Slow b u t gradual i m p r o v e m e n t in general condition a n d blood picture. T h i s is a typical case of t h e s e c o n d stage of s p r u e (VAN DER BURO) in w h i c h t h e cardinal s y m p t o m s are p r e s e n t b u t t h e h~emolysis a n d blood d e s t r u c t i o n proceeded apace. CASE III. Male, 48. Civil Servant. M a r r i e d . B o r n in E n g l a n d . T w e n t y - n i n e years ago w e n t to C h i n a , J a p a n a n d Korea. T w e n t y - f i v e to t w e n t y - o n e years ago in E n g l a n d . F o r last t w e n t y - o n e years in Hong Kong. Personal History.--Malaria, 1907 to 1910 a n d in 1921 ; d y s e n t e r y (? variety) in 1910 a n d 1920. I n 1921, on s t e a m e r , gradual o n s e t o f diarrhoea, in m o r n i n g s , two or t h r e e loose stools b e c o m i n g white, frothy, b u l k y a n d offensive ; t o n g u e a n d m o u t h sore at this t i m e ; lost weight rapidly ; p r e s e n t weight, 60 kg., diarrhoea increased to seven stools daily b y 1925, w h e n ( N o v e m b e r ) blood a n d m u c u s a p p e a r e d for a s h o r t t i m e ; anaemia b e g a n to be severe ; h a d to give u p work a n d return to E n g l a n d . Condition on Admission (Sth April, 1 9 2 6 ) . - - E m a c i a t e d a n d e x t r e m e l y pale. Examination.--Shin, earthy t i n t a n d inelastic ; tongue, clean, b u t glazed a n d w i t h p r o m i n e n t " s h a v e n " f u n g i f o r m papillae, was n o t sore ; teeth, poor ; throat, clear ; lungs a n d heart, clear ; abdomen, wall t h i n a n d flaccid ; s u b c u t a n e o u s fat a b s e n t ; distension of lower half, m o r e pron o u n c e d before m o t i o n s passed ; liver, d i m i n i s h e d to h a n d ' s b r e a d t h in r i g h t m a m m a r y line ; splee~t, n o t enlarged ; sigmoid colon, t h i c k e n e d a n d palpable ; legs, s h o w old p i g m e n t e d scars of varicose ulcers ; central nervous system, clear ; urine, 1008, acid, faint trace a l b u m i n o n 9th April, 1926, n o n e since 16th April ; no sugar, a n d n o t h i n g pathological n o t e d in d e p o s i t ; stool, no ova nor protozoa seen ; m u c u s present, pale, greasy, offensive, f o r m e d a n d often constipated. T h e constipated stools were definitely b r o w n in colour. Blood.--9th April, 1926, red blood corpuscles, 2,000,000 ; h~emoglobin, 60 p e r cent. ; w h i t e blood corpuscles, 5 , 2 0 0 ; colour i n d e x , 1"5; p o l y m o r p h o n u c l e a r n e u t r o p h i l e s , 72 p e r cent.
296
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
lymphocytes, 26 per cent. ;. large mononuclears, 2 per cent. ; poikilocytosis, anisocytosis, and one normoblast seen ; no paramtes present. O n 27th April, red blood corpuscles, 3,200,000 ; heemoglobin, 70 per cent. ; eolour index, 1"1 ; Wassermann reaction, negative. Course.--Slow i m p r o v e m e n t in general condition, with rapid i m p r o v e m e n t in appearance of stool. T r e a t e d in bed with diet. D u r i n g period of observation diet was 6 pints of milk, 2 ozs. rusks, 4 ozs. sago pudding, and one egg daily. T h i s is a second case of long-continued sprue w h e r e the blood destruction had become profound ; in some respects like Case I I , b u t the process had been m o r e gradual and less severe. T h e blood picture resembled Addisonian anmmia, b u t clinically the case was typically s p r u e without marked alimentary s y m p t o m s .
V.--TABULATED
RESULTS.
CASE I--F~ECES,
Weight of Stool (in grammes).
Days.
Total Urobilin and Urobilinogen (in milligrammes).
1st
...
2nd 3rd 4th 5th 6th 7th
... ... ... ... ... ...
219 574 222 182 235 215 275
9o 138 196 98 288 193 259
Total
...
1,922
1,262
Urobilinogen (in milligrammes of Urobilin).
N o t estimated. J 81 205 142 166
I
Average pe~ day
274"5
594
180
148'5
Percentage present as Urobilinogen = 71. Quantity of Urobilin per 100 g. fmces = 67"5 rag. Estimations of urine not made, as qualitative tests uniformly negative. CASE II--F~CE$.
Days.
Weight of Stool (in grammes),
Total Urobilin and Urobilinogen (in milhgrammes).
Urobilinogen (in milllgrammes of Urobilin).
115 245
185 537
128 518
Xst
...
2nd 3rd 4th 5th 6th 7th
... ... ... ... ... ...
614 17
1,326 9
1,229 7
611
745
687
Total
...
1,602
2,802
2,569
229
400
367
Average per day
Percentage present as Urobilinogen = 91"75. Quantity of Urobilln per 100 g. fieces ~ 174"7 rag.
SOME OBSERVATIONS ON U R O B I L I N
OUTPUT
IN SPRUE
297
CASE. I I - - U R I N E .
Day s .
Q u a n t i t y in cubic centimetres.
Urobilin ix milligrammes.
Schlesinger's Test.
Ehrlich's Test.
++ + ++ ++ ÷ + +
1st 2nd 3rd 4 th 5 th 6th 7th
... ... ... ... ... ... ...
1,930 1,400 2,125 2,555 1,870 1,540 2,330
9 4 8 15 2
÷ + + + + weak +
Total
...
13,750
41
--
__
1,964
6
--
--
A v e r a g e p e r day
2 1
T o t a l d a i l y e x c r e t i o n of U r o b i l i n = 406 rag.
CASE III--FfiECES.
W e i g h t of Stool in g r a m m e s ) .
Days.
Total Urobilin and U r o b i l i n o g e n (in r n i l l i g r a m m e s of U r o b i l i n ) ....
I
1st
.,.
2nd 3rd 4th 5th 6th
... ... ... ... ...
347 165 220 182 140 184
TotaJ
...
1,238
:
U r o b i l i n o g e n (in m i l l i g r a m m e s of Urobilin).
454 158 306 227 254 306
286 154 174 178 195 179
1,705
1,166
284
194
I A v e r a g e p e r day
206'3
P e r c e n t a g e p r e s e n t as U r o b i l i n o g e n = 68. Q u a n t i t y of U r o b i l i n per 100 g. f~eces = 137"7 m g .
298
SOME OBSERVATIONS ON UROBII.IN OUTPUT IN SPRUE
CASE III--1)IIINE.
Q u a n t i t y l n cubic centimetres,
Days.
U r o b i l i n in milligrammes.
1st
...
2nd 3rd 4th 5th 6th
... ... ... ... ...
2,300 2,500 1,720 2,120 2,775 1,920
2"4 1'6 0'8 2'0 2"5 2"8
Total
...
13,335
12"1
Averag e p er day
2,222'5
Ehrlich's Test.
÷ ÷
Schlesinger's Test.
÷
weak
+ ÷ +
÷ ÷
2"0
T o t a l daily excretion of U r o b i l i n = 286 mg.
DISCUSSION.
In the past, many statements have been made with regard to the cause of the colour of sprue stools. Most of these explanations have taken for granted that the absence of the normal colour had to be accounted for by showing the bile-pigment products to be present in an altered form. VAN DER SCHEER (1905) states that except for traces of unreduced bilirubin, all the bile-pigment is present as leuko-urobilin (NENCKI). DOLD (1924) repeated this statement, held a substance responsible by the same name and defined it as a colourless chromogen of urobilin. In English text-books on Tropical Medicine the substance is mentioned, with occasional changes of name to leucobilin in which form it is even harder to recognise. Now, although not uncommon in German text-books of physiology as a clinical term, none of these names figure in modern English or American physiological chemistry works, and their use leads some students to consider that in sprue one has to deal wlth a new and peculiar product of hepatic (or enteric) activity. This opinion, indeed, is shared by some authorities on the disease, who invariably quote SCHMIDT'S corrosive sublimate test as a proof of the presence of bile-pigment products. M A N S O N - B A H R brought forward interesting features in his work in Ceylon, and showed that fresh sprue stools did not give a positive Schmidt's reaction until some time had elapsed. In a footnote, he quotes STRASBURGERas maintaining the identity of NENCKI'S leucourobilin and the substance ordinarily known as urobilinogen.
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
9@9
On referring to the figures obtained in the three cases studied, it will be seen that :-1. In Case I, without severe anmmia, the quantity of total urobilin present (t80 mg.) is only slightly higher than the average normal (140 mg.) (LICHTENSTEIN, 1924 ; CHARNAS, 1909) ; but of this total 71 per cent. is a colourless compound, and the remaining 29 per cent. is distributed throughout a stool of average daily weight 274"5 g., which is double the normal. Hence, the dilution of the actual pigment is approximately six times. 2. In Case II, whilst the average daily weight of f~eces is only one and a half times the normal, the quantity of total urobilin is much increased. The more interesting feature is that the percentage of pigment present as the chromogen is 91"75 ; hence, the actual dilution of pigment is eighteen times. LICHTENSTEIN, from his series of observations, states that the quantity of urobilin excreted in 100 g. of f~eces seldom exceeds 120 rag. ; which figure compares with the 174"7 mg. in this case, in spite of the fact that the total quantity of f~eces per diem is simultaneously increased. 3. In Case III we have an intermediate stage, in which more of the nrobilinogen has passed over into the oxidised state than in Cases I and II. The proportion of urobilinogen, however, is high (68 per cent.). The dilution is much less--about four times--thus agreeing with the appearance of the stool. Here, then, we have figures showing that the ratio of urobilinogen to urobilin is very high in sprue, and that the actual amount is much increased in those cases with anaemia. As the form in which the chromogen is present allows it to be measured by a urobilinogen reaction, the suggestion is made that, in future, leucobilin and similar synonyms be avoided and the more familiar term urobilinogen used. It is not proposed to assert that this is the complete explanation of the colour of sprue stools, since the well-known experiment of HERMANN must be kept in mind. This matter of the Hermann isolated loop of intestine and its secreted f~eces is one of great difficulty ; STARLINGstates "After some weeks an isolated loop (of intestine with full blood supply) will be found to contain a semi-solid material similar to fa~ces in appearance, consistence and chemical constitution," on which experiment is based the statenlent in many physiology books, that the normal colour of f~eces has no relation to the presence of bilepigments and their products. SCHMIDT and STRASBURGER,however, quote EHRENTHAL thus : Hermannsche Ringkot ungefarbt, grau aussieht. In this uncertain position it is as well to remember that, in sprue, we h~,ve a proof of the effect of bile-pigment products in producing a colour approaching normal in the stool, for, as has long been known to clinicians, the greyish stool of sprue gradually assumes a brownish tint when exposed to light and air, a change due to the oxidation of urobitinogen to urobilin.
800
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
VI.--FURTHER EXPERIMENTS. In order to confirm these high ratios of urobilinogen to urobilin, further experiments were performed ; two on fresh specimens of sprue stool (within half an hour of their being passed) and two, on consecutive days, on a normal patient on whom a plastic surgical operation had been p e r f o r m e d . SPRUE CASE A, Chronic type ; firm, pasty stool, Wasting and previous history of sore tongue ; anemia ; red blood corpuscles, 2,800,000 ; hmmoglobin, 65 per cent. ; weight of stool, 128"28 g. ; urobilinogen, 215 rag. ; total urobilin and urobilinogen, 235 mg. ; urobilin, 20 rag. Urine gave negative Schlesinger and very weak Ehrlich. In this case, 91"5 per cent. of the bile-pigment product in a fresh sprue stool was present as urobilinogen. SPRUE CASEB. One and a half years' duration ; fluid, slightly pigmented, frothy stools ; n o t m u c h wasting ;
tongue symptoms very slight ; liver diminished in size ; red blood corpuscles, 4,700,000 ; hmmoglobin, 85 per cent. ; weight of stool, 101'98 g. ; urobilinogen, 78 rag. ; total urobilin and urobilinogen, 104 mg. In this case, 75 per cent. of the product was present as the colourless chromogen. SURGICALCASE. Stool normal in colour and appearance. On milk, fish, bread, etc. First Day.--Weight of stool, 84"25 g. ; urobilinogen, 84 rag. ; total urobilin and urobilinogen, 165 mg. Second Day.--Weight of stool, 58 g. ; urobilinogen, 27 rag. ; total urobilin and urobilinogen, 98 mg. Daily Average.--Weight of stool, 71 g. ; urobilinogen, 55"5 rag. ; total urobilin and urobilinogen, 131"5 rag. In this case, 42'2 per cent. of the product was present as urobilinogen.
Unfortunately. there are no other figures available of this percentage in fresh, normal stools. V I I . - - T H E RELATION OF UROBILIN OUTPUT TO BLOOD BREAKDOWN. T h e total urobilin o u t p u t was first employed by EPPINGER (1920) to obtain a measure of the rapidity of blood destruction, but he made use of a factor which he stated to represent the normal a m o u n t of bilirubin secreted per diem. This factor he had obtained by carefully weighing the material obtained from two patients with complete bile fistulm. T h e theoretical figure thus obtained did not, however, coincide with those obtained in practice. T o obviate this, LICHTENSTEIN (1924) has introduced an " index of blood destruction which he defines as " the rapidity with which a patient destroys his blood compared with that of a normal person." T o obtain this index, LICHTENSTEIN argues thus : " T h e weight of the patient in kg. is obtained easily ; m o d e r n figures agree that approximately one-twentieth of this weight is blood. Of the blood, 14 per cent. by weight normally is h~emoglobin and 4"47 per cent. of the h~emoglobin is h~ematin. T h u s , the total quantity of h~ematin in any person can be calculated if their weight be known, and can be adjusted more accurately if the percentage of hmmoglobin in the blood be estimated in a reliable manner. At the same time, the average daily a m o u n t of urobilin excreted can be measured, and from this figure the equivalent amount of h~ematin per day calculated. "
SOME OBSERVATIONS ON UROBILIN
OUTPUT
IN SPRUE
301
T h u s , i n a m a n of 70 kg. w e i g h t , the total h~ematin i n t h e b l o o d c a n be c a l c u l a t e d as 21"9 g. I f t h e u r o b i l i n e x c r e t e d p e r d i e m be t h e n o r m a l 150 m g . (LICHTENSTEIN), t h e n this is calculated to b e d e r i v e d f r o m 153 m g . of h~ematin. T h u s all the h~ematin c o u l d b e d e s t r o y e d i n a b o u t 140 days. A p p l y i n g this to t h e f o r e g o i n g cases : CASE I. Body weight, 62"7 kg. ; hzemoglobin, 85 per cent. ; weight of blood, 3,135 g. ; weight of h~emoglobin, 0"85 × 0"14: × 3,135, or 373 g. ; weight of h~ematin, 0"044:7 × 373, or 16"673 g. ; average daily output of urobilin, 183 rag. ; equivalent to ~0 × 183 or 186"7 rag. of hmmatin. Therefore, the time taken is ~ . , ~ X 1 or 89 days. The corresponding time for a normal man of the same weight, with 100 per cent. of h~emoglobin, and excreting 150 mg. of urobilin per diem, is 128 days. Therefore, the index is l~s or 1"4. That is, he is using his blood 1"4 times as rapidly as a normal man. CASE I I .
Body weight, 52"75 kg. ; hmmoglobin, 55 per cent. ; weight of blood, 2,637"5 g. ; weight of hmmoglobin, 0"55 × 0"14 × 2,637"5, or 203"088 g. ; weight of hmmatin, 9"078 g. ; average daily output of urobilin, 406 rag. ; equivalent to ~xo x 406 or 414 mg. of hmmatin. Therefore, the time taken is 0~7~ or 22 days, approximately. The corresponding time for a normal man of the same weight, with 100 per cent. of hmmoglobin and excreting 150 rag. of urobilin per diem, is 108 days. Therefore the index is ½0s or 4"9. CASE III.
Body weight, 60 kg. ; h~emoglobin, 70 per cent. ; weight of blood, 3,000 g. ; weight of h~emoglobin, 0"7 × 0'14 × 3,000, or 294 g. ; weight of hmmatin, 294 × 0'0447 or 13"142 g. ; average daily output of urobilin, 286 rag. -, equivalent to ~'~l × 286 or 291"7 mg. of hmmatin. Therefore, the time taken is a~#{ or 45 days, approximately. The corresponding time for a normal man is I22'7 days. Therefore, the index is -~.ea~ or 2"7. VIII.--SUMMARY. 1. E s t i m a t i o n s of b i l e - p i g m e n t p r o d u c t s b y the m e t h o d of T~RWEN, have b e e n carried o u t o n t h r e e cases of s p r u e , i n each case e s t i m a t i o n s b e i n g c o n t i n u e d daily for o n e week. 2. I n two cases w i t h severe anaemia t h e average daily o u t p u t o f " u r o b i l i n '~ ( u r i n e a n d f~eces) was m u c h a b o v e n o r m a l . I n t h e case w i t h o u t m a r k e d anaemia, the o u t p u t was w i t h i n t h e l i m i t s of n o r m a l . 3. T h e p e r c e n t a g e of t h e total p r o d u c t s p r e s e n t as u r o b i l i n o g e n - - t h e colourless p r e c u r s o r of u r o b i l i n - - v a r i e d f r o m 68 to 91"75, a n d these figures were c o n f i r m e d i n fresh stools f r o m two f u r t h e r s p r u e p a t i e n t s . 4. T h e p e r c e n t a g e of t h e total of t h e bile p i g m e n t p r o d u c t s p r e s e n t as u r o b i l i n o g e n i n a c o n t r o l surgical case was 42"2. 5. T h e i n d e x of b l o o d d e s t r u c t i o n (LICHTENSTEIN) has b e e n w o r k e d o u t o n t h e t h r e e m a i n cases a n d the results s h o w t h a t t h e y b r e a k d o w n t h e i r b l o o d 1"4, 4"9, a n d 2"7 t i m e s as r a p i d l y as the n o r m a l case is c a l c u l a t e d to do. 6. T h e s u g g e s t i o n is m a d e t h a t the t e r m s l e u c o - u r o b i l i n a n d l e u c o b i l i n , as at p r e s e n t u s e d , b e r e p l a c e d b y the t e r m u r o b i l i n o g e n , as the s u b s t a n c e p r e s e n t i n t h e f~eces i n s p r u e gives all the characteristic r e a c t i o n s of this g r o u p .
809.
SOME OBSERVATIONS ON UROBILIN OUTPUT IN SPRUE
In conclusion I wish to thank Drs. G. C. L o w and P. H. MANSON-BAHR for access to their cases in the Hospital for Tropical Diseases, W.C.1, and to the Resident Staff at the Hospital for their whole-hearted co-operation. To Dr. H. B. NEWHAM, C.M.G., I am indebted for permission to work in his laboratory and for his ready help and guidance at all stages of the work.
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