Vol. 37 FUNCTION OF PANTOTHENATE IN BACTERIA 425

REFERENCES

Barron, E. S. G. & Lyman, C. M. [1939]. J. biol. Chem.127, 143.

- Lipton, M. A. & Goldinger, J. M. [1941]. J. biot.Chem. 141, 957.

Berkman, S., Dorfman, A. & Koser, S. A. [1942]. J. Bact.43, 6.

Dickens, F. & 8imer, F. [1931]. Biochem. J. 25, 973.Dixon, M. [1934]. Manometric Methods. Cambridge:

University Press.Dorfman, A., Berkman, S. & Koser, S. A. [1942]. J. biol.

Chem. 144, 393.Edson, N. L. [1935]. Biochem. J. 29, 2082.Fenton, H. J. H. & Jones, H. 0. [1900]. J. chem. Soc. 77, 77.Fildes, P. [1940]. Brit. J. exp. Path. 21, 67.

& Richardson, G. M. [1937]. Brit. J. exp. Path.18, 292.

Gladstone, G. P. [1937]. Brit. J. exp. Path. 18, 322.- [J939]. Brit. J. exp. Path. 20, 189.

Greville, G. D. [1939]. Biochem. J. 33, 718.Hills, G. M. [1941]. Chem. Ind. 60, 220.Krebs, H. A. [1937a]. Biochem. J. 31, 661.

[1937 b]. Biochem. J. 31, 2095.& Eggleston, L. V. [1940]. Biochem. J. 34, 1383.& Henseleit, A. [1932]. Hoppe-Seyl. Z. 210, 33.

Lipmann, F. [1939]. Nature, Lond., 143, 436.

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Biol., N.Y., 43, 151.Pilgrim, F. J., Axelrod, A. E. & Elvehjem, C. A. [1942].

J. biol. Chem. 145, 237.Pratt, E. F. & Williams, R. J. [1939]. J. gen. Phy8iol.

22, 637.Quastel, J. H. & Webley, D. M. [1941]. Biochem. J. 35, 192.Smyth, D. H. [1940]. Biochem. J. 34, 1598.Still, J. L. [1941]. Biochem. J. 35, 380.Teague, P. C. & Williams, R. J. [1942]. J. gen. Physiol.

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Mosher, W. A. & Rohrmann, E. [1936]. Biochem. J.30, 2036.

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Wohl, A. & Claussner, P. [1907]. Ber. dtsch. chem. Ge8.40, 2308.& Oesterlin, C. [1901]. Ber. dt8ch. chem. Ges. 34, 1139.

Woolley, D.-W. [1941]. J. biol. Chem. 140, 311.

Micro-method for Estimating Vitamin A by the Carr-Price ReactionBY H. HOCH (Freedom Research Scholar), From the Haymead8 Emergency Ho8pital, Bi8hop'8 Stortford,

and the Hale Clinical Laboratory, London Hospital

(Received 16 March 1943)

In the micro-determination here described an at-tempt has been made to eliminate errors due to

H F E DCBC A

LI- - 450mm; - -~-* 450mm;. .............-

Fig. 1. Photographic arrangement. A, light source, two100 W. coiled coil bulbs. B, space for filters: (1) 'Calorex'2 mm.; (2) 'Signal green' 1 mm. (manuf. by -MessrsChance, 10 Princes St, S.W. 1). C, diffusing glass 2 mm.D, reaction tube and standard tubes. E, W-ratten 26 redgelatin filter. F, enlarging lens at suitable distance togive image of tubes of about natural size. G, slit, 3-5 mm.wide, in front of slide which is moved in vertical direction.H, slide.

fading, differences in the colour of test and standard,and interference by non-specific absorption. The

tube containing the reaction mixture is photo-graphed in light of a selected wave-length rangetogether with a series of standardt tubes of equaldimensions (see Fig. 1),-and the photographic filmis analysed by means of photoelectric photometry.The advantages of this method are: (1) the workingrange is between 0-06 and 0-4 i.u. vitamin A in asample; such small amounts cannot be determinedby photoelectric colorimetry in the usual way, butit is necessary to work in this range when makingserial investigations on the blood of small animalsand is convenient in investigations on humanbeings; (2) rapid mixing is ensured as a consequenceof the small volume of the reacting liquids (0.02 ml.);(3) a record of the colour produced can be obtainedas early as 2 sec. after the initiation of the reaction,and its course can be followed photographicallywith accurate timing, thus avoiding the lag ofgalvanometer readings in photoelectric methods[Dann & -Evelyn, 1938; Yudkin, 1941]; (4) thereadings are independent of fluctuations in the lightsource during the reaction.

27-2

426 H. HOCHA detailed study of the error due to fading in the

Carr-Price reaction is presented below. The prin-ciple of the method is generally applicable tomeasurement of colour in small volumes.

EXPERIMENTALDetermination of vitamin A

Standards. Vitamin A. Any stable substancewhich has an absorption maximum in the region of620 m,u is suitable as a standard. Solutions ofcuprammonium sulphate in water were preparedranging from 2- 145 to 0

ESTIMATION OF VITAMIN Atype was mounted at the upper end of the microscope.By moving the film under the microscope perpendicularlyto the slit, galvanometer readings were obtained alternatelyfor maximal transmission through the film (correspondingto maximal light absorption in a tube) and for the back-ground of the film. The differences between the readings ofthe maxima and the background were plotted against theconcentration of CuSO4 in the standards, and the unknownvalue was read from the line connecting the values for thestandards. This approximates to a straight line under theoonditions used (Fig. 2). It is essential that the backgroundbe evenly illuminated. Photometry ofthree or four adjacenttubes in the range of tho unknown concentration is suffi-cient. Fig. 3a shows the calibration line obtained withvitamin A-naphthoate. The arachis oil containing the

The fading error in the Carr-Price reactionIt is generally accepted that the maximum colour

of the Carr-Price reaction is proportional to thevitamin A present. Spectrographic examinationconfirms this view. Notevarp & Weedon [1936]showed that the blue colour obeys Beer's law.McFarlane & Sutherland [1938] achieved excellentagreement (average error 1 %,) between the twomethods in analyses of fish-liver oil concentrates,but the error was greater with the-oils themselves(average error 6%). Jensen & With [1939], ana-

50

10 20 30 40 sec. 50(a)

vO) 10 20(b)

30 40 sec. 50

100;- 100cS0 C

E 50

:0h'4.,0 0 10 20 sec.(c,)

c;z0

0

K1 =0'8D K2=0.0 10 sec.

(f)

501-

~ 10 20 sec.(d)-

K1=0-8K2=0-02

0 10 20 sec.(g)

(e)100 -

50 -K,= 1-2K20-05

0 10 20 sec.- (h)

Fig. 4. Fading of Carr-Price colour given by different preparations with vitamin A activity. (a) Vitamin A-naphthoate:(D 0 380 i.u. (16-7 i.u./ml.); 0-283 i.u. (12-9 i.u./ml.). (b) P-Carotene: 0-77/Ag. (35.5,ug./ml.). (c) Blood extract:0-135 i.u. vitamin A (5-55 i.u./ml.); 0-143,ug. carotene (5-9jug./ml.). (d) Blood extract with low concentration ofvitamin A and high concentration of carotenoids: 0-054 i.u. vitamin A (2.9 i.u./ml.); 0-337,ug. carotene (16-7,ug./ml.).(e) Rat-liver extract: 0-264 i.u. vitamin A (13-0 i.u./ml.); 0-041,tg. carotene (2-0,ug./ml.). (f), (g) and (h): calculatedfading curves.

vitamin A-naphthoate was diluted with light petroleumand aliquots were treated as described for extracts. Theequation of the calibration line was found to be

y= 18.25x - 1-03,

x being the concentration of CuSO4.5H20 as a percentageand y the coilcentration of vitamin A in i.u. The standarddeviation was i 3.3% in the range of 4-22 i.u./ml. Withconcentrations approaching 2 i.u./ml., corresponding to0-04 i.u. in 0-02 ml. of reaction mixture, the error graduallyrises to 15%.

lysing livers of mammals, birds and man, give thevalues found forB"S 61/El 328m/h as 2-70, 2;63anA 2-75 respectively, with a=0-404, 0-436 and0-406, where B1 %. S61 represents the extinctioncoefficient of the Carr-Price reaction mixture (lightfilter S 61) and El % 328 the extinction coefficientfor light of wave length 32AmtL.

Variable fading rates have been observed byDann & Evelyn [1938] not to affect the maximumcolour intensity; and Meunier & Raoul [1938]

Vol. 37 427

100

unl

500

Ca

0c003

Uo

I UU r- In,4r--

v r

428 H. HOCHsuggest that they may serve as additional criteriafor distinguishing vitamin A from vitamin A2.No information could be found concerning colour

development during the first j-min. after the startof the SbCl3 reaction. This was studied by themethod described above in samples of pure vitaminA-naphthoate and ,B-carotene, and in extracts ofhuman blood and rat liver. If the maximum colouris reached before fading starts, these curves (Fig. 4)indicate a variable onset of the latter and variablerates of fading. If, however, fading begins as soonas the coloure-d compound is present, i.e. at thetime of adding the reagent, the ideal maximum isnever reached, but its value could be calculated byapplying a correction, derived from the fading rate,to the observed maximum.The calculations are based on a first order type

reaction curve both for colour development and forfading in the neighbourhood of the maximum. Thefading rate is assumed to be proportional to theconcentrations of the molecules of vitamin A whichhad reacted with the SbCl3, giving the blue colour.The relation between observed blue colour, ex-pressed as percentage of the maximum colour as-suming that no fading has taken place, and thetime, is given by

1OOx kk (e-kit _k2taO k2.-kl t -

the solution of the differential equationdx/dt = aokle kLt _ k2x;

where x = concentration of vitamin A as chromo-genic compound, ao= concentration of vitamin A atthe beginning of the reaction, k, = reaction constantof the colour development, k2 = reaction constantof the fading reaction, t= time in sec.The values of the ordinates corresponding to a

series of finite exposures as used in these experi-ments (1j and 1 sec.) were calculated from

10 t2'xdt 1 / 1 1- 2aO h_tj k2-kl -k +k2 ItL

Fig. 4 shows curves (f, g and h) calculated by meansof this formula with varying values for k, and k2,in which the ordinates represent the values for100x/ao expressed as percentage of the highest valuefor lOOx/ao obtained.

It is possible to estimate k2 directly from theslopes of the experimental curves at their points ofinflexion after the maximum, when colour forma-tion has practically stopped. From this figure andthe value of the ordinate obtained at the firstreading, k, can be determined by the formula, andwith it, theoretically, the time ofthe true maximum.

k2 was 0-018 0-003 for vitamin A-naphthoateand 0-005 for carotene. Wide variation from 0-003to 0-1 was observed with extracts from various

I943sources. The value of the first ordinate in 55 experi-ments ranged from 90 to 100% of the maximumvalue found. The accuracy of the determination of k1was limited by the practical difficulty of ensuringcomplete mixing within 2 sec.When k, is taken as 0-7 and k2 as 0-01, the first

ordinate works out at 88-5% of the maximum.Where kL is 0-7, or greater, and k2 is 001, or greater,the position of the maximum according to the aboveformula must be at or earlier than 6 sec. afterreaction. The positions of all maxima were foundto fulfil this condition.

In dilutions below 4 i.u. vitamin A/ml. the curvesoften deviate from the calculated, the fading con-stant being greater at the beginning than wouldcorrespond to the value found at 22 sec. afterreaction. The effect was not detected with concen-trations above 4 i.u.Iml. or with solutions ofvitamin A-naphthoate. It may be caused by inter-fering substances in the blood. Agreement is ob-tained between curves calculated with k,= 0-8 andexperimental curves for concentrations greater than4 i.u./ml., but the validity of the formula derivedneeds further test on accoAunt oftheuncertainty of kc .Table 1. The estimation of vitamin A and caro-

tenoid8 and the fading rate of the Carr-Price colourwith two concentrations

SubjectI.V.

M.

B.

A.B.

J.R.M.

R.B.

D.M.

J.R.M.

W.

Vol. ofreactionmixture

mi.0-01990-02130-01840-01950-01770-0189'0-0190-02130-01870-02160-0210-01950-01870-01860-0210-01960-01770-021

Conc. in bloodper 100 ml.serum or

Conc. in plasma Serumreaction(- A ormixture pg. plasmai.u. vit. i.u. caro- usedA/ml. vit. A tene ml.2-1 51 57 0-0813-6 49 55 0-1561-24 25 32 0 092-59 28 31 0-182-67 53 64 0 094*50 47 73 0-184-37 103 90 0-0817'52 103 90 0-1565-27 97 78 0-1027-24 87 83 0-182-92 76 97 00816-3 78 95 0-1566.52 135 87 0 09

12-7 131 89 0-184-12 85 90 0-1027.55 82 89 0-182-66 52 74 0-093-64 43 78 0*18

% fadingfor

vit. A at22 sec.after

reaction4533

24-318-25.58

43327

11-714-5

The observation that the percentage fading mea-sured by the value at 22 sec. in two different con-centrations of an individual extract is nearly thesame (Table 1), may be taken as evidence of a firstorder type of fading curve. Whether the mechanismof the fading reaction is unimolecular or whetherit is bimolecular with a second constituent in excesswas not investigated.

Vol. 37 ESTIMATION OF VITAMIN A 429It is of practical interest to know-whether correc-

tions shoVld be applied to the maximum colourvalue observed in samples that fade at differentrates. Table 2 gives the calculated maximum values

Table 2. Calculated correction8 to maxima, reached with different value8 of k, and k2k2 0 005 0.010002 0*03 0050 0.1

k1 Max. Corr. f22 Max. Corr. f22 Max. Corr. f22 Max. Corr. f22 Max. Corr. f22 Max. Corr. f220-8 97*2 - 6 5.1 94.5 -3.5 14*0 90 9 +0*5 27-5 87.6 +4 38-7 82*9 +10 57-2 73-4 +24.5 82*71*0 97-7 -5 5-9 95*3 -2-5 15.1 92-3 +05 28-9 89-4 +4 404 84-5 +10 58-5 76X6 +21.5 83.9

Max. = calculated maximum value of colour as percentage of theoretical total, without fading.Corr. =corrections to experimental maximum values as percentage.f22 =fading 22 sec. after reaction as percentage of the maximum.

as percentages of the theoretical values withoutfading (1st columns), and the corresponding correc-tions for samples whose constants of fading differfrom those of vitamin A-naphthoate (2nd columns).The 3rd columns give the percentages of fading22 sec. after reaction.The distribution of the constants of fading in

33 blood extracts is given below. These figures werecalculated from-the values for the blue colour dueto the vitamin A,. and it was assumed, though notquite correctly, that the yellow colour of the ex-tracts was entirely due to ,B-carotene. Possible differ-ences in fading rate of the blue colour formed withfl-carotene with various extracts were disregarded:

k2 < 0-005 0-005- 0-01- 0-02- 0-03- > 0-040-01 0-02 0-03 0-04No. of 2 4 11. 14 2 '0samples 'The corrections for the majority of extracts from

blood are within + 5% (see Table 2), and so theyneed not be applied for practical purposes, but withfading rates greater than k2 = 0-05 the observedmaxima yield values which are appreciably toolow, and corrections are necessary.The correctionfor p-carotene in the Carr-Price reactionThe blue colour given by carotene with the Carr-

Price reagent was measured in the same way as

that described for vitanxin A (Fig. 3b). Values forconcentrations lower than 8,ug. carotene/ml. weretoo small to be measured accurately. For the pur-pose of correcting the Carr-Price values of extracts

containing carotene the calibration line was extra-polated graphically to reach the point where thecalibration line for vitamin A crosses the abscissa,and this value, 0-056% CuS04. 5H20, was sub-tracted from all figures of the calibration curve forcarotene. The reason why the calibration line forvitamin A does not reach the zero point may be asmaller light transmission of the control comparedwith that.of water.

SUMMARY1. A photographic device for the measurement

of the Carr-Price reaction is described.2. The rates of fading with different extracts

were studied, and the corrections which it is neces-sary to apply to the values obtained were deter-mined.

3. If the concentration of vitamin A in the bloodis calculated from the observed maximum colour'without correction for fading the error will generallynot be greater than 5 -

I wish to thank the authorities of the Haymeads Emer-gency Hospital, Bishop's Stortford, and of the LondonHospital for the facilities which made this work possible andthe British Drug Houses, Ltd. for preparations of vitamin Aand carotene.

REFERENCES,

Dann, W. J. & Evelyn, K. A. [1938]. Biochem. J. 32,1008.

Jensen, H. B. & With, T. K. [1939]. Biochem. J. 33, 1771.McFarlane, W. D. & Sutherland, A. J. [1938]. Canad. J.

Re8. B, 16, 421.

Meunier, P. & Raoul, Y. [1938]. C.R. Acad. Sci., Pari8,206, 1148.

Notevarp, 0. & Weedon, H. W. [1936]. Biochem. J.30, 1705.

Yudkin, S. [1941]. Biochem. J. 35, 551. --