604 BIOCHIMICA ET BIOPHYSIC.k .\CT.k VOL.

2 4 (i957)

Q U A N T I T A T I V E E S T I M A T I O N OF S I A L I C A C I D S

II. A C O L O R I M E T R I C R E S O R C I N O L - H Y D R O C H L O R I C ACID M E T H O D ' , *"

LARS SVENNERHOLM
Department of Medical Biochemistry, University o[ Gothenburg, Gothenburg (Sweden)

In a previous communication 1 the quantitative estimation of sialic acids with orcinol-

hydrochloric acid (Bial's reagent) has been described. With this method the keto-
hexoses, when tested in dilute solutions, gave a colour that was impossible to dis-
tinguish from that of the sialic acids. Thus Bial's reagent cannot be used for the
estimation of sialic acids when they occur together with ketohexoses. The amount of
ketohexoses is low in animal materials and will not seriously disturb the determination
of sialic acids. SPRINGER it, however, has reported that sialic acids also occur in plant
materials, where the content of ketosugars is much greater. Therefore, a systematical
investigation was performed to find out whether it would be possible to determine
sialic acids when they occur in the same samples as ketohexoses. Reagents used for
the estimation of ketohexoses were tested in order to obtain a reagent that gave
different colours with ketohexoses and sialic acids. The results of the testing are
collected in Table I.
In the resorcinol method of COLE et al. ~ sialic acids and fructose gave quite
different colours. From the other tests some general trends in the colour formation
of sialic acids could be observed. The colour formation was strongly promoted by
Cu ~+ or Fe ~+, while alcohols or acetic acid inhibited it to a great extent. On the basis
of these general principles a method was worked out. The molar absorbancies of sialic
acids and other sugars in the new method were determined and compared to those
obtained with Bial's reagent. Mixtures of N-acetylsialic acid and other carbohydrates
were analysed by dichromatic readings and the amount of the former was calculated.
Some applications of the method on biological materials are mentioned.

EXPERIMENTAL
Materials
Sialic acids. N,O-diacetylsialic acid (CxsH,INO10,/H,O/) f r o m b o v i n e s u b m a x i l l a r y m u c i n a n d
N-giycolylsialic acid (CllH1,NOxo) f r o m pig s u b m a x i l l a r y m u c i n were a gift f r o m Professor
G. BLIX, U p p s a l a . N-acetylsialic acid (CllH19NOg) was p r e p a r e d b y t h e m e t h o d described b y
t h e a u t h o r TM.
Other carbohydrates. G l u c o h e p t u l o s e w a s a gift f r o m Professor N. K. I:~ICHTMEYER, B e t h e s d a ,
a n d d e s o x y r i b o s e a gift f r o m ProfesSor S. LALAND, Oslo. All t h e o t h e r c a r b o h y d r a t e s were c o m -
mercial p r e p a r a t i o n s . T h e y were all recrystallized before use.
Resorcinol (Merck) w a s u s e d w i t h o u t f u r t h e r purification.
* A p r e l i m i n a r y r e p o r t h a s a p p e a r e d in Biochem. J., 64 (1956) i IP.
* Q u a n t i t a t i v e E s t i m a t i o n of Sialic Acids I, ref. 1
Re]erences p. 6rL
VOL. 24 (I957) QUANTITATIVEESTIMATION OF SIALIC ACIDS I I 605

TABLE I
T H E B E H A V I O U R OF N ~ A C E T Y L S I A L I C ACID A N D FRUCTOSE I N COLORIMETRIC TESTS D E V I S E D
FOR T H E E S T I M A T I O N OF KETOSES

The intensity of the colour f o r m e d is g r a d u a t e d f r o m + to + + + .

Colour development
Methods N-A cetylsialic acid Fructose
1oo I~g 50 I~g

ROE (1934) 8 None Orange

Resorcinol-HCl-ethanol + + +
RoE et al. (1949) 4 None Orange-red
Resorcinol-HCl-ethanol-thiourea + + +
COLE et al. (1954) ~ Blue-violet Orange
Resorcinol-HCl-glycerol-Cu ~+ + + + + + +
KULKA (1956) 6 Red Orange
Resorcinol-HCl-ethanol-Fe 3+ + + + +
Present method Blue-violet Orange
Resorcinol-HC1-Cu ~+ + + + + + +
POGELL (1954) 7 None Violet
Skatol-HAc-ethanol + + +
P A T M A L N I E K S AND G A R D E L L ( 1 9 5 6 ) 8 None Red
Thymol-HAc-ethanol + +
DISCHE AND BORENFREUND (1951) 9 None Violet
Cysteine-carbazol-H2SO 4 + + +
V A N C R E V E L D (1927) l° Red-violet Blue
Diphenylamine-HCl-ethanol + + + +
DlSCHE (193o) 11 Violet Blue
Diphenylamine-H2SO4-HAc + + + + + +

Hydrochloric acid (Merck) AnalaR, density 1.19, a t least 36.4 % HC1. Fe 3+ n o t m o r e t h a n

O.OOOI ~0.
o . I M copper sulphate. 24.97 g of CuSO 4, 5 H~O was dissolved in distilled w a t e r to I 1.
A myl alcohol. I 1 of isoamyl alcohol w a s mixed w i t h 200 ml of concentrated hydrochloric
acid and left for a week. The alcohol was w a s h e d w i t h 2oo ml of w a t e r a b o u t ten times a n d dried
w i t h a n h y d r o u s p o t a s s i u m c a r b o n a t e a n d distilled. The fraction with a b.p. of I3O-I33° C was
used.
Resorcinol reagent. 2 g of resorcinol w a s dissolved in IOO ml of distilled w a t e r (stable for
m o n t h s in a refrigerator). IO ml of this solution w a s added to 80 ml of c o n c e n t r a t e d hydrochloric
acid containing 0.25 ml of o.i M copper sulphate. The v o l u m e of the reagent w a s m a d e up to
ioo ml w i t h distilled water. The r e a g e n t w a s p r e p a r e d at least 4 h o u r s before use. I t w a s stable
for a week w h e n stored in the refrigerator.
Blank reagent, I t h a d the s a m e composition as the resorcinol r e a g e n t b u t contained no
resorcinol.

Procedure
2 m l of samples containing lO-3O/~g of N-acetylsialic acid or a corresponding a m o u n t
of other sialic acids, were pipetted into three tubes. 2 ml of resorcinol reagent were
added to t w o of the tubes (test samples) and 2 m l of blank reagent to the third tube
(sample blank). In the same manner standard solutions of o, 15 and 3o ~g N - a c e t y l -
sialic acid were prepared. If the u n k n o w n samples also contained other carbohydrates,
a m i x t u r e of these carbohydrates were run as standards at t w o different concen-
tration levels. The t u b e s were heated for 15 m i n u t e s at IiO ° C in a n oil b a t h or in
Re[erences p. 6zx.
606 ~. SVENNERHOLM VOL. 24 (1957)

a boiling water bath. After heating, the tubes were cooled in running water, 5 ml
of amyl alcohol were added, the tubes were shaken vigorously and then placed in
ice water for 15 minutes. The contents were transferred to centrifuge tubes and spun
at 5OO-lOOO r.p.m, for one minute. The tubes were placed in ice water again and left
there until the photometrical reading was taken. The amyl alcohol phase was trans-
ferred to 5o m m microcells with ballooned pipettes and read at 450 and 580 m/, in
a Beckman Spectrophotometer model B against pure amyl alcohol. The readings were
completed within one hour after heating.
The absorbancy of the blank sample was subtracted from the mean of the test
samples and the amount of N-acetylsialic acid calculated by means of the equations
derived from the standard solutions of N-acetylsialic acid and the carbohydrate
mixture.

RESULTS

Optimal conditions/or colour production

Composition o/the reagent. In the orcinol-hydrochloric acid reaction 1 a final concen-
tration of about 5 N acid was found optimal. As it was permissible to mix aliquots
of sample and reagent, IO N hydrochloric acid was used in the reagent. The amount
of resorcinol used (0.2 g/Ioo ml reagent) was also the same as the amount of orcinol
used. The optimal concentration of Cu ~+ was determined b y analysing the absorbancy
of IO and 40 t~g N-acetylsialic acid. Maximum absorbancy was achieved with 0.20-
0.25 ml of Cu~+-solution/ioo ml reagent. When no oxidants were added to the reagent
the absorption curves of N-acetylsialic acid and fructose were much lower and the
curve shape of N-acetylsialic acid quite unspecific (Fig. I).
As the method was intended for the quantitative estimation of sialic acid in
biological materials, including lipid extracts, it was necessary to eliminate the influ-
ence of f a t t y materials. Lipids cause an opalescence of the samples, which can be
removed b y the addition of organic solvents. Addition of miscible alcohols before
heating strongly diminished the colour development; the depressing effect of mono-
alcohols was the largest, of dialcohols less, and of glycerol least. The colouring m a t t e r
was therefore taken up in amyl alcohol after heating. The colour then turned more
blue and the absorption m a x i m u m shifted from 57 ° mt~ to 580 m/~. The same phe-
nomenon was also seen when the colouring matter in the orcinol-hydrochloric acid
reaction was extracted with the alcohol.
Heating conditions. The testing with the orcinol-hydrochloric acid reagent re-
vealed that the reproducibility was better when lipid-bound sialic acid was estimated
at a somewhat higher temperature than at ioo ° C. The cause of this is still unknown
but a better mixing of the rather inhomogeneous samples occurs at the higher
temperature. The heating of the samples has therefore been performed at two temper-
atures, at IiO ° C in an oil bath, and in a strongly boiling water bath. In the former
case closed tubes (Thunberg tubes) were used, in the latter, however, centrifuge tubes.
At the higher temperature the m a x i m u m absorbancy of N-acetylsialic acid was
achieved b y heating for I5 minutes. When the heating was performed for the same
length of time in a strongly boiling water b a t h the absorbancy was about lO% lower.
The absorbancy, however, increased with a prolonged heating time at least up to
30 minutes, but the absorbancy of hexose increased still more (Table II). To eliminate
Re/erences p. 6zz.
VOL. 2 4 (I957) QUANTITATIVE ESTIMATION OF SIALIC ACIDS I I 607
the influence of hexoses a short heating time would have been desirable, but in that
event quantitative values were not obtained, since the colour development was
slower for bound than for free sialic acid. Heating for 15 minutes in a boiling water
bath is the minimum time recommended for the investigation of protein-bound sialic
acid. For lipid extracts and more exact determinations heating at IiO ° is preferable.
T A B L E II
THE RELATIONSHIP BETWEEN ABSORBANCY AND HEATING TIME

T h e h e a t i n g w a s p e r f o r m e d in a boiling w a t e r b a t h . T h e a b s o r b a n c i e s a f t e r h e a t i n g a t i i o °
for i5 m i n u t e s are also given.

H e a t i n g at I o o ° C H e a t i n g at z x o ° C
Materials
z 5 rain 20 rain 25 rain 3 ° rain z 5 rain

N-Acetylsialic acid 0.899 1.o45 1.137 1.2oo 1.o45

(34.64/~g)
Galactose o.199 o.318 0.383 o.436 0.325
(500/~g)
Ribose o.332 o.477 0.469 o.475 o.475
(50/~g)

Stability o/the colour/ormed. When the samples were stored at room temperature
the absorbancy diminished by about 5% during the first hour. After this initial
decrease, the absorbancy diminished much less rapidly (I %/hour) during the following
three hours. The decrease in absorbancy was, however, negligible if the tubes were
stored in ice water until the reading was taken.
Sample blank. In order to compensate for non-specific colour in biological
materials a sample was run with blank reagent (without resorcinol), The sample blank
was subtracted from the test sample before calculation of the sialic acid amount.

1.2 /2/5 ~, 0.6 t, ~ ' , ,

o
0.8 ~
I~ \ :0.40.5

0.6 0.3 t

\
O/ 0.2
0.:' 0.1
400 500 600 700 400 500 600 700
WavelengthmF Wavelength rnp,
Fig. I. T h e a b s o r p t i o n s p e c t r a of N-acetylsialic Fig. 2. T h e a b s o r p t i o n s p e c t r a of [ ] - - [ ] 20.48
acid a n d fructose w i t h a n d w i t h o u t Cu ~+ in t h e F g N-acetylsialic acid; @ - - @ 23.00/~g N,O-
reagent. W i t h o u t Cu 2+ : ® - - ® 25/~g, fructose ; diacetylsialic acid; . . . . . 19.68/zg N-glycolyl-
[ ] - - ~ 5 ° #g, N-acetylsialic acid. W i t h Cu 2+. sialic acid.
Q - - O 25/~g, f r u c t o s e ; I I - - I I 5 ° #g, N - a c e t y l -
sialic acid.
Re]erences p. 6zz.
 608 L. SVPLNNERHOLM VOL. 24 (I957)

Application o/ the method to sialic acids and other carbohydrates

Sialic acids. Three naturally-occurring sialic acids are known, all with the same basic
structure 13. They have tentatively been named N-acetylsialic acid (CllHloNO,),
N,O-diacetylsialic acid (C13H21NOlo, H20 ) and N-glycolylsialic acid (CnH~gNOlo) 12.
Several other names for the same substances have been used by other researchers;
this has been discussed in a recent paperL
The absorbancy indices of the three different acids were determined. As can be
seen in Table I I I the molar absorbancy index (AM) of N-glycolyMalic acid was 2o %
higher than the indices of the other two. The same difference was also seen in Bial's
reaction. In comparison with the indices found with the latter method, the absorbancy
indices with the resorcinol reagent were about 5o% higher. Beer's law was valid up
to an absorbancy of 1.5 when 5o mm microcells were used. The same absorbancy
coefficients were found if the readings were performed in IO mm cells. The method
was also tested with greater concentrations of sialic acids in the samples. When IO mm
cells were used, there was a departure from the linearity for absorbancies greater
than o.5, amounting to 7.5% at an absorbancy of o. 9.

T A B L E III
T H E MOLAR ABSORBANCY I N D I C E S ( A M ) OF SIALIC ACIDS AND O T H E R CARBOHYDRATES AT 5 8 0 n l / l
IN T H E RESORCINOL R E A C T I O N

For comparison, the molar absorbancy indices of the other c o m p o u n d s are also given in percentage
of t h e index of N-acetylsialic acid (CllH1,NOg). Further, the s a m e d a t a for the orcinol-hydro-
chloric acid reaction are tabulated.

Resorcinol reaction Orcinol reaction

Heating at too ° C Heating at xzo ~C Heating at zo6 C

Materials Colour
Percentage o/ AM Percentage o/ AM Percentage o/
AM CnH19NOs CnHlgNOs Cl aH19N09

N - A c e t y l s i a l i c acid Blue 8o2o ioo 952o lOO 5895 ioo

N,O- D i a c e t y l s i a l i c acid Blue 7980 99.5 9498 99.8 5798 98.4
N - G l y c o l y l s i a l i c acid Blue 9545 119.o 11280 118. 5 7o60 119.7
Galactose Yellow 48 0.6 74 0.8 S5 i .4
Glucose Yellow 59 0-7 86 o.9 97 i .6
Mannose Yellow 67 o.8 i 15 1.2 131 2.2
Arabinose Red-violet 2o7 2.6 567 6 600 io
Ribose Red-violet 996 12.4 I395 14.7 t 554 26.4
Glucuronolactone Red-violet 278 3-5 53 ° 5-6 585 9.9
Desoxyribose Olive green 953 11.9 938 9.9 442 7.5
Fructose Yellow 1544 19.2 166o 17. 4 16o2 27.2
Glucoheptulose Blue-green 295 ° 36.8 3465 36.0 705 ° 12o
L-FUcose Red-violet 28 o.35 38 0. 4 53 0-9
2-Deoxyglucose Yellow-red 1417 17.7 1634 17.2 -- --
Digitoxose Red 2920 36.4 333 ° 34.5 444 ° 75

The absorption spectra of the acids were also estimated and showed a good
agreement between 500-700 mtL (Fig. 2). The differences in the curve-shapes seen
at shorter wavelengths m a y depend on an admixture of small amounts of other carbo-
hydrates, since sugars forming fuffural and furfurol analogues have an optimum
around 450 m/,. The method is thus also a sensitive indicator of small amounts of
contaminating sugars in a sialic acid preparation.
Re[erences p. 6xz.
VOL. 9.4 (1957) QUANTITATIVE ESTIMATION OF SIALIC ACIDS I I 6o9

Other carbohydrates. Several other carbohydrates were also tested and their molar
absorbancy indices calculated and compared to that of N-acetylsialic acid. The
determinations were performed after heating at both IOO° and IiO ° C for 15 minutes.
Ketosugars and 2-deoxy sugars had somewhat greater percentage absorbancy when
heated at the lower temperature, while the aldosugars had considerably greater
percentage absorbancy at the higher temperature. The aldohexoses and 6-deoxy-
hexoses had very low absorbancy indices and in rather great amounts only caused
a minor error. The absorbancies of the ketohexoses were still high but their influence
was eliminated b y reading the samples at a second wavelength. Pentoses, glucuronic
acid, 2-deoxyglucose and digitoxose had in addition to the maximum absorbancy
around 450 m/~, a second maximum at 580 mF. This is obvious from Fig. 3 in which
the absorption spectra of N-acetylsialic acid and some other carbohydrates are shown.
The method is thus unsuitable for samples containing pentoses, glucuronic acid and
2-deoxyhexoses; in those cases the diphenylamine method of DISCHEn is to be pre-
ferred.

SIALIC ACID F RUC'I'OSE GALACTOSE- MANNOSE I RIBOSE

1.0 0.02 mg 0.O1 m, O.1 rng 0.1 m g
o

.io
<
0.8 I I

f t
/./'~ !
0.6

i
i ~
~ !
/ I
i !~Ji
] 'k"l
/ /
I
0.~ /f'x.j,~
!i i i
i\
, !
/ ~
0.2
/
~..J
I
'\
\
'\
\
\.
x,
\ "\

I I I I I I I I
400 500 600 400 500 600 400 500 600 400 500 600 400 500 6 0 0
Wavelength mp
Fig. 3. T h e a b s o r p t i o n s p e c t r a of N-acetylsialic acid a n d o t h e r c a r b o h y d r a t e s .

Mixtures o/N-acetylsialic acid and other carbohydrates

Since biological materials besides sialic acids contain other carbohydrates, mixtures
of N-acetylsialic acid and galactose, and N-acetylsialic acid and fructose were
analysed with the resorcinol reagent. The samples were read at 450 and 580 m/~ and
the concentration of N-acetylsialic acid calculated from simultaneous equations. The
recovery of N-acetylsialic acid from known mixtures is shown in Tables IV and ¥.
If mixtures of other carbohydrates interfere with the determinations a correction
can be made, provided that the approximate composition of this mixture is
known 14.
R*levences p. 6Ix.
61o L. SVENNERHOLM VOI_ 24 (1957)
Sensitivity and accuracy
The molar absorbancy of N-acetylsialic acid, calculated on the amyl alcohol phase,
was 8,ooo and 9,50o for 15 minutes heating in a boiling water bath and oil bath of
IiO ° C, respectively. This means that the absorbancy of i/~g of N-acety!sialic acid
is about 0.o30. The sensitivity of the new method is 50% greater than that of the
orcinol method.
The standard deviation calculated from 20 samples of N-acetylsialic acid, each
containing 34.64/~g of the acid, was ~ 0.8% when heated at IiO ° C and i 1.o%
when heated at IOO° C. The standard deviation was in general twice as great in
biological materials.
The absorbancies of the standards have been very constant during the year that
the method has been employed, so that standards are not necessary in each run if
the conditions applied are strictly followed.
TABLE IV
DETERMINATION OF SIALIC ACID IN MIXTURES WITH GALACTOSE BY DICHROMATIC READINGS
T h e s o l u t i o n s w e r e h e a t e d a t lOO ° C f o r 15 m i n u t e s in o p e n t u b e s . T h e a b s o r b a n c i e s w e r e d e t e r -
m i n e d in a B e c k m a n S p e c t r o p h o t o m e t e r m o d e l B w i t h a s l i t - w i d t h o f 0 . 3 - 0 . 4 r a m , 5 ° m m cells.
The readings were done against a reagent blank.

N-A cetylsialic acid

Absorbancy
N-A cetylsialic acid Gala,close found* recovered
I*g ltg 450 mp 580 rot* I~g (%)

34.64 o 0.295 0.875 34.64 --

25.98 ioo 0.398 0.668 25.67 99
17.32 20o 0.34 ° 0.490 17.62 lO2
8.66 300 0.320 0.275 8.60 ioo
o 400 0.355 0.083 -- --

* T h e c a l c u l a t i o n s w e r e d o n e f r o m e q u a t i o n s derived f r o m t h e first a n d l a s t s a m p l e s .

TABLE V
DETERMINATION OF SIALIC ACID IN MIXTURES WITH FRUCTOSE BY DICHROMATIC READINGS
T h e s o l u t i o n s w e r e h e a t e d a t lOO ° C f o r 15 m i n u t e s in a b o i l i n g w a t e r bath. T h e a b s o r b a n c i e . ,
were determined in a Beckman Spectrophotometer model B with a slit-width of 0.3-0. 4 mm
T h e r e a d i n g s w e r e done a g a i n s t a m y l a l c o h o l .

N-Acetyl sialic acid

Absovbancy
N-A carl sialic acid Fructose ]ound* recovered
ttg fig 45 ° m # 580 mlt pg (%)

o o 0.072 O.Ol 4 -- ---

21.44 o 0.258 0.558 21.44 --
16.o8 5.oo 0,539 0.485 16.39 lO2
lO,72 io.oo 0,774 o.39o lO.74 too
5.36 15.oo i.OlO 0.305 5.51 lO 3
o 20.00 1.255 o.215 -- --

* T h e c a l c u l a t i o n s w e r e d o n e f r o m e q u a t i o n s derived f r o m t h e first and l a s t s a m p l e s .

Application o~ the method/or biological samples

The method has been employed in a number of analytical problems:
(a) Quantitative estimation of N-acetylsialic acid in serum, milk a n d cerebro
References p. 61I.
VOL. 2 4 (1957) QUANTITATIVE ESTIMATION OF SIALIC ACIDS I I 611

spinal fluid and in methanol-chloroform extracts of nervous tissue 14. (b) Study of the
release of N-acetylsialic acid from mucoproteins and gangliosides 14. (c) Analysis of
the effluent from anion-exchange columns in the isolation of N-acetylsialic acid from
different sources TM.
For the assay of protein-bound N-acetylsialic acid in body fluids, besides sialic
acid standards, equimolar solutions of mannose-galactose were used as standards.
It was found that 92-98% of the absorbancy at 580 mt~ was due to N-acetylsialic
acid, if the heating conditions were IOO° C for 15 minutes. No significant difference
was seen between samples from normal and pathological cases. A rather accurate
and rapid determination of sialic acid in, for example, serum proteins can thus be
done only by reading the samples at 580 m/z and afterwards correcting the values
by multiplying by o.95.

ACKNOWLEDGEMENTS

I wish to thank Miss MARGIT S J 6 S T R A N D for valuable technical assistance. The work
was supported in part by a grant from the State Foundation for Promotion of Medical
Research in Sweden.

SUMMARY

A n e w m e t h o d for t h e q u a n t i t a t i v e d e t e r m i n a t i o n of sialic acids is described. O p t i m a l c o n d i t i o n s

for t h e colour f o r m a t i o n were i n v e s t i g a t e d . T h e m o l a r a b s o r b a n c y indices of N-acetylsialic acid,
N-glycolylsialic a n d N , O - d i a c e t y l s i a l i c acid a n d s e v e r a l n a t u r a l l y - o c c u r r i n g c a r b o h y d r a t e s were
d e t e r m i n e d . T h e m e t h o d is a b o u t 5 ° % m o r e s e n s i t i v e t h a n t h e orcinol-hydrochloric acid m e t h o d
g e n e r a l l y u s e d . T h e influence of o t h e r c a r b o h y d r a t e s is also c o n s i d e r a b l y lower w i t h t h e resorcin
r e a g e n t . T h e r e c o v e r y of N-acetylsialic acid f r o m k n o w n m i x t u r e s w i t h g a l a c t o s e a n d fructose
w a s d e t e r m i n e d . T h e d e t e r m i n a t i o n of sialie acid in b o d y fluid p r o t e i n s is briefly discussed.

REFERENCES

1 L. SVENN~RHOLM, Arkiv Kemi, Io (1957) 577.

2 G. F. SPRINGER, Naturwiss., 2 (1955) 37.
3 j . H. RoE, J. Biol. Chem., lO 7 (1934) 15.
4 j . H. RoE, J. H . EPSTEIN AND N. P. GOLDSTEIN, J. Biol. Chem., 178 (1949) 839.
5 COLE, HANES, JACKSON AND LOUGHMAN, cited b y D. J. BELL in Moderne Methoden der Pltanzen-
analyse, Vol. 2, S p r i n g e r Verlag, Berlin, 1955, p. 21.
6 R. G. KULKA, Biochem. J., 63 (1955) 542.
7 B. M. POGELL, J. Biol. Chem., 211 (1954) 143.
8 j . PATMALNIEKS AND S. GARDELL, Scan& J. Lab. Clin. Invest., 8 (1956) 223.
Z. DlSCHE AND E. BORENFREUND, J. Biol. Chem., 192 (1951) 583 .
10 S. VAN CREVELD, IKlin. Wochschr., 6 (1927) 697.
11 Z. DISCHE, Mihrochemie, 8 (193o) 4.
12 L. SVENNERHOLM, Acta Soc. Med. Upsaliensis, 61 (1956) 75.
18 G. BLIX, E. LINOBERG, L. ODIN AND I. WERNER, Nature, 175 (1955) 34 o.
14 L. SVENNERHOLM (in p r e p a r a t i o n ) .

Received December 2oth, 1956