l'rom the Biochemical Department of the Medical Sobcl Institute. Stockholm.

A ~hloriniet~riciil ('i~i~boii-monoside-heluo~lo~)ill Mebtiod of I)etrenniniition for Clinical USC.

1:y

KARL-GUSTAV PAUL and HUGO THEORELL.

(Rewired 4 311ue 1942.~

Carhon niotiosiclc poihoitiiig in on(' sliape or aiiotltt>r is on(' of t I I P coiiinionrst forms of poisoning - perhaps. indeed. vv(w t hr wniinonest. &i larqe nuni lm of c :~r l~o~i- i i ioi io~i~t~~-l ie i i ioglol~i i~ iiie t h o t l s ( ) t (1 e t erininn t ion for c li n ica 1 us(' 11 a vc t h ere f ore 1 )w n worked out. of which the colorimetric iiiethods have Iwcn thosc. itiost usrti. ( R A Y . BLAIR ;~iitl THOMAS (1932). I ,~wz(*zAI< (1936).

MAY (1939 a and 1)) arid HEILRIEYER (1933)).

HbCO method of deterininntion. 1 t must

11.4Y (1937). OETTEL (1938 i L &lid 1)). XI.4TTHES alld GROSS (19:%<)).

Certain tleinnnds must be iitade of ;I good colorinic+ric. clitlicikl

I ) Iw c~asy to perforni: 2 ) be practicable a t any 1iospit:il a t :ill without calling f o r

:I ny pa rticularl y elaborate extra apparatiir: 3) prct'eri~bly be R micro-iiiethod so tha t saiiiples ~ a y I)(>

iakcii Froiii the t ip of the finger or the lobe of the car :ind vciioi~s puncture may be avoided.

The nii~joritv of iiiethods fulfil th r al,ove rcquircnielits. .is appe:irs froin fig. 1. however. :I certain amount of CO is

dissociated fro111 the CO-hemoglohin when the blood is diluted, and the rnlrie ohtainccl will thus be too low. -411 the iiicthods listttl in the refrrcnccs work \\ i th strongly clilutetl blood. Thr authors have accordingly workrtl out a niethorl in which this source of crror i q avoided by iiieaiis of inakiiig colorimetric dc- terminations 011 t h e hlootl in very thin cuvettes. it heing thus

286 KARL-GUSTAV PAUL AXD HUGO THEORELL.

possible to dilute so slightly tha t the error arising from the di- lution may be obviated. ,4s reading-instrument a Stufen-photo- meter may be used. as i t will he found in most larger hospitals. Greater accuracy could, naturally, be achieved by using a spect- ral photonieter instead. And i t is of course easy to ada.pt t,he method for this apparatus if required.

I

100 3 D U Y O 0 ’mdi I. I 00

Pig. 1 . Curve 1: HbC‘O-saturated blood is diluted up to 500 times: Na,S,O, i n atlded, folloaetl by R colorimetric determination (curve 2) . Curve 3 shows the

same thing. bat with renewed 1~iibt)ling with CO aftrr the dilution.

The pri t lc iple: The pigments occurring in the blood - Hb, HbCO, HbO, and possibly MetHb are translated with reducing agents to two --- Hb and HbCO. The percentage of HbCO in total-Hb can then easily he determined by Heilniayer’s elabo- ration of Vierordt‘s method (HEILMEYER, 19.33). According to the expression:

1 1

where X = the desircd %-content of the one pigment. in this case HbCO.

concentration extinction coefficient

A = absorption-relation = - for HbCO at

t,he wave-length 1,.

COLORIMETRIC.4L C A R B O K - M O ~ O Y I D E - I I I ~ O ~ L O ~ I ~ METIIOD. 287

concentration ~-

extinction coefficient ii = absorption-relation =

the u-ave-length A,. concentration

extinction coefficient I3 = absorption-relation = ~~

wave-length 1,. concentration

extinction coefficient 1) = absorption-relati~~n =

wa ve-Imgth A‘.

for HhCO a t

for Hh a t the

for Hh at the

El E,

(2 =-, where F:, and E, represent the extinctions a t A , and

2,. we can draw a curve with <’,-content HbCO and Q on t h t b

Thc~ extinctions for Hb and HbCO are deterniined in a cuvette that will be described below. The thickness of the layer is thus constant. As, further, [Hb] and [HbCO] are of equal magnitude, since the smiples have been taken from the same experimental

s u l ) j w t and equally diluted, we can substitute for

axes.

1 1 1 A’ fi and

The greater the difference l~etween and QHb, the inore iiccurwte will be the result. An investigation of the values that WARIIIJR(:. CHRISTIAN and NEGELEIN obtained spectro-photo- metrically gives the following: the ]!-band for HhCO lies a t 420 inrr, for H b at 430 nip. These values arc combined with the low absorption-values at alternatively e. g. 510 and 530 nyr. (Stufcn-filters S 61 and S 53 are generally available).

The values of p (the “molar ahsorption-coefficient”) are piveil niiiItipIi~d hy 1 O P

HbCO Hb

2 .72 (),12 = 22.!)?

28s KARL-(;US'L'AV PAUL A X D l l U 0 0 TIIHOHELL.

'l'litl greatest difference between the quotients for HM'O an(l HI) is t h u s obtained by cniploying t h e wave-lengths 430 ni;r i111tI 530 nip. True. these figures were ohtained with spectro- pliotoiiieter values; hut our cleterniinations show the condition3 for the Stiiftw-photometer to be ahout the sanie. Thc most suit- able filters in tlic Rtufen-photoiueter iire tlius S 43 and S 53. c.orrcsporitling to A, and respectively in the forniulii.

nil. in ;my casc not over 0 . 1 nil . Froiii the first division to thih second the voliiiiic is twice ;is great. t h u s nlmut 0.16 n i l . O n ( ~ thus ohtains il clilutioii of tht. hlootl in the ratio 1: 3. Thr. t i p of thp pipttc. ~iiiist Iw very f i n c h . An outer di:mictrr of O.(i- 0.h

i i i i i i . seems to he suitnblc. I f it i\ l i i r p r the out-flowing fluid ivill IN, unnecessarily exposed to thv air.

The cuvette (fig. 3) consists ot two p l ~ ~ ~ i ~ - p i ~ r a l l ~ l . polislied glasb slides, 25 x 35 \ ' 2 nini.. separated hy two bands ot cold-rolled and tempered steel of f;xtory-controlletl

. The most suitable thick- ness has I w n found to br 0.1o m u . I f o i i c uses O.(i:< - - o . o A i i in i . it i h .

wrtninly. possihh~ to carry orit cdorinictric tlctcrniiii;ttioiis on untlilutetl blood, Lilt with this thickness we found it difficult to get const:iiit valurs For Q,,,, (on w couple of occasions with cstreniely low values of Q - ahout 4 . 2 0 instead of l . h ? oiic ~ u l d st(. \vct~B ;ll)sorf,tio~i-l)i11ids from HbO, in the spectroscope). The glass s1idt.s are provided a t the top with twin 1)evelling tha t f o r m ii little groove into which the t ip of t h e pipette i h

inserted so tha t there is the least possible exposure to the air. The "cuvclttc" is held together with two ordinary scre\\--claiiip~ castd 111 rubher.

F I ~ . :<. h ~ c t l o l l ( ~ t ( ( I \ c . t t t * .

('0LOKIMETRIC.ZL C A l ~ ~ 0 ~ - M l ~ ~ ~ J ~ I ~ ~ - H ~ ~ M 0 ~ ~ 1 ~ 0 1 ~ 1 ~ M E T I I O D . 28!1

Ai siiiiilar technique i lh regards cuvettes has c~irlier 1)ceii eni- plOYf'(1 bp KLT'lTF:RFIEI,I) (1912).'

diluting fluid WI' usetl:

Sodiuni citr,itc. 2 i i ( 1 . . . . . . . . . . . . . . . . . . . O.:i g Saponin . . . . . . . . . . . . . . . . . . . . . . . . . 0.9 g

Ihffrr (4 volh. m/15 prim. 4 6 vols. ni/15 s w . phohpI ia t (~ . l)FI 6.117) Sodiuni hydro-hulphitr. 2 ;iq . . . . . . . . . . . . . . . 0.; g

to 1 0 . 0 i d .

T l i c solution is I)uffcred in order to prevent SO, forriietl by oxitiiition of Nii,M20, from sinking tlie pH too inuch, which woultl ineaii t ha t .stroriiatin might he precipitated.

I t ma~7 1ic.r~ he iiientionetl t h a t the smile tubes tliilt arch iisctl tor I)lood-tcsts in alcohol-clcteriiiinations ( "Widlliiirk tubes") ( W I ~ M A R K 192%) may lw usctl for sninplcs for carhoii nionoxidc tletcriiii~iatioiis. Such n tube holds alwut 0 . 1 2 1111. The blood is sucked over in t h e pipette via a ccintinirter-long rubber ligature. The taking of saiiiples and sending of saiiie t o the lalmratories can be effected in precisely the s i tme way in both cases. ;I point t h a t i s prol)al)ly of value froni the psychological point, of view. iis it has sonietiines proved troul)lesonic to get blood-tests from itiotorists in case of traffic.-accidents v-hcrr the infliimccb of alco- hol is suspected.

.Ur,thod: From tlie t ip ot the finger, the lolw of the ear or ii

*~\Vidniark tube" hlood is sucked up t o the first division, tlilut- ing fluid t o tlic second division, after which the liquids itre mixed by sucking I)itck\~:irds i l ~ ~ d for\\.iirds bet\vcrn t he two 'bulbs' of t h o pipette. \Vlicn t h r t ip is then placed in the previously nientionctl groove, the solution is sucked in to ;L capillary layer whcrr it is protected froin air and froni drying. Es4:3 and Es,,,

h4:{ .

HS5? ;ire then cleterniinecl. the quotient is fornictl and the clcsircvl

value is obtained froni the curve.

I t is iiitwvstiiig to note that BUTTERFIELIJ, without giving any rrason for this. rlors not IISV ii lcssrr tliliitioii of t h blood than 1 : 3, altliougli he tricd t o work with as i.oncrntratrtl solutioiis as possi1)les.

290 KARL-OUSTAV P.4UL A X D I€U(;O TIlEOlLELL.

Results. H b

EhJ8 ES63 Q 1 . 2 6 7 0 . 2 6 0 4 . ~ 7 1 . 2 5 4 0 . 2 0 1 4 . 8 1 1 . 2 5 0 0 . 2 6 2 4 . 7 7

Average: 1 . 2 5 7 0 . 2 fi 1 4 . 8 2

HbCO

0 . 7 4 7 0.367 2 . 0 4 0 . 7 4 9 0 . 3 6 3 2 . 0 3 0 . 7 6 0 0 . 3 7 0 2 . 0 5

Average: 0 . 7 .i 2 0. :I f i 7 2 . 0.i

E 543 %61 Q

HbCO was prepared here and in the following experiments in the same way: a mixture of 49 vols. of N, and 1 vol. of CO, prepared in the ordinary way from formic acid, was bubbled through blood for 20 minutes. By using diluted carbon mono- xide the effect of physically dissolved CO was avoided in the experiments described in the following. Experinients with blood from different healthy experimental subjects have given the same result. On the basis of these values a table was made up with assumed, varying values of Q .

Q 4 . 8 2 4 . 6 0 4 . 4 0 4 . 2 0 4 . 0 0 3.80 3.60 3 . 4 0 3 . 2 0 3 . 0 0 2 . s 0 2 . 6 0 2 . 4 0 2 . 2 0 2 . 0 5

n;-contcnt of HbCO 0 5 . 5

11 17 23 29 36 43 50 58 66 74 83 92

100

Control experiment: The HbC'O-content was determined in samples with a known proportion o € HhCO. HbCO was prepared in the way already dercribed.

COLORIMETRICAL CARBON-MOKOXIDE-HEMOGLOBIX METIIOD. 9:) I

cc HbCO- cc HbO,- solut.

0 . 0 5 0 . 0 3 0 . 0 5

0.10 0 . 2 0 0 . 1 0

0 . 1 0 0. 1 2 5 0.15

0 . 1 5 0 . 1 0 0 . 2 0 0 . 2 0 0 . 2 0 0 . 2 5

0 . 5 0 0 . 3 0 0. 3 d

0 . 4 5 0. 4 0

sotut . 0.43 0 . 4 5 0 . 4 3

0 .40 0 . 8 0 0 . 4 0

0 . 4 0 0 . 3 7 5 0 . 3 5

0.3 5

0 . 2 0 0 . 3 0

0 . 3 0 0 . 3 0

0.25 0 . 5 0 0 . 2 0 0. 1 3 0 . 1 0 0 . 0 5

s4.i E

1.29 1 . 2 8 1 . 3 0

1 . 2 0 1 .2s 1 . 1 6 1 . 3 8 1 . 3 4 l . 2 e 1 . 1 5 1 . 1 6 1 .2 '1 1.2s 1.11 1.01;

1 . 2 u 1 . 0 s

1.11;

1 . 0 5 0 . 9 8

lcq

0 . 2 9 2 0 . 2 9 0

0. 3 0 0 0 . 3 0 0

0 . 2 8 2 0 . 3 4 6

0 . 3 4 0 0 . 3 4 :3 0 . 2 9 5

0 .310 0.3Afi 0 . 3 7 2 0 . 3 1 4 0 . 3 2 4

0 . 3 1 7 0 . 3 i 1 0 . 4 3 3 0. 3 9 5

0 . 4 7 i

. 53

0 . 3 1 6

ESqx E 5

L 67

4 . 4 2

4 . 4 1 4 . 3 3

4 . 0 0 4 . 0 3

4.11 3,9!l 3 . 9 4 3. 7 ti 3 . 9 0 3 . 7 4 3 . 3 i 3 . 4 4 3 . 5 4

3.27 3.17 2.!IO 2 . 6 7 2.4 ii 2 . 2 0

CRIC.yb

10 10 10 20 20 20 20 25 30 30 3 3 . 3

40 40 40 50 50 60 70 80 90

M:e also carried out deterniinat.ions parallel

Empir.7;

1 0 . 5 1 0 . 5

13 23 2 1 . 5

20 23. 5

2 5 . 5

31 2 6 . 5 3 1 . 5

44 41.3 38 48 51.3 61 67.5 8 0 . 3

92. 5

I jeviat.

+ 0 . 5 + 0.5

+ 3 t 3 + 1 . 3

0 + 3 . 5 + 0.5

-t 1 3.5

-- 1.s + 4 + 1 . 5

- 2 . .- 2 + 1 . 3

+ 1 ~~- 2 . 5

+ 0 . 5

+ 2 . 5

with a niet)hod described hy PAIJL and WRETLIND (1942), based upoii preci- pitation of the reduced 1ienioglol)iii under certain conditions, while HbCO remains in solution.

Accord. ES4:, %.~ 9.1 Y "L HM'O .,,, K. tvRET,,IN,) Difference

2 . 2 3 0 . 2 6 5 4 . 6 4 4 . 3 7 2 . 3 1 . 2 4 0 . 2 6 ~ ; 4 . 6 6 4 ti 2.0 1 . 0 2 0 . 2 5 0 4.0s 20.5 23 2 . 5 0 . 9 9 0 . 2 4 1 4 .07 21 27 f i 1 . 0 8 0 . 2 ' 1 2 3 . 9 i 2 4 . 5 23 1 . 5

Serum from a jaundice patient with ~ ~ e u l ~ n ~ r a c h t - v a l r i ~ 1 : 60 gave S,, = 0.oa and S,, = 0.01. blood-sample from a patient with chronic niyelosis, fully able to work and coniplaining only of a slight tenderness in the abdomen, and with 64 O O Hb, 3 . 4 niill. red and 22.5.000 white blood corpuscles per cubic niillinieter gave QHb = 4 . h 6 and QHbCO =: 2 . 0 ~ . One may thiu abstract from these possible sources of error.

Summary.

('iirl)oii-ii~oiioaide-helieiiio~lol)i~i iiicthods of t1ctcrtiiin;itioii that t l ~ i i l with strongly diluted h1oo:I must give too lo\\- values owing t o thv tiict that cnr l~o~i- i i ior ioxicl~-~iei~io~loI~in dissociates on tli- l i i t ioti . .\ biniple n,cthod. sufficiently accurittc for clinical usti,

has I)et,ii \\orkrtl out from I\ hich tliesr sources of error lia\-c hern c~liminiitctl.

'rhc d~.tcr~ii i i i i l t iol~ IS easy t o carry out a d tiikes only a f ( . \ t

tiiinutes. .\n itIiportiint advantage of the iiiethod is t ha t i t enahlei detrr1iiiri;itioii on :I micro-scale, so that blootl iiiay lw taken froni t h r t ip of the finger. Samples can be sent i i i capi1l:try tubes. ;ih for itist:irice is ttoiic in t l ie ciISe of 1)Iood-trsts for alcohol iic- cor,liiig t o \l'ic!niark's nicthod.