Determination of plasma progesterone during pregnancy

CLINICA CHIMICA ACTA

DETERMINATION

OF PLASMA

DURING

PROGESTERONE

PREGNANCY

H.J.VAN Department

913

DER

MOLEN

ofObstetrics and Gynecology, State University, Utrecht (The Netherlands) *

(Received January 10th.1963)

SUMMARY

A modification of SHORT'S method for the determination of plasma progesterone is described, which allows the estimation of 0.5-1.0,ugper sample. The reliability of the method is tested and plasma levels in cord and peripheral blood during pregnancy are reported.

INTRODUCTION

For clinical purposes the determination of the urinary excretion of Q-pregnane3u, zoa-diol has long been used to obtain an impression of the endogenous progesterone production. In the last few years, however, increased attention has been paid to the determination of progesterone itself in blood or plasma. For reviews and details of the methods used for the determination of plasma progesterone, we refer to the recent articles by PEARLMAN', SHORTY and ZANDER~. The present investigation was stimulated by the publication of the plasma progesterone determination as used by OERTEL et aL4, who published for the first time plasma progesterone levels during the menstrual cycle. These results led us to seek a method that .might allow the determination of plasma progesterone levels during the menstrual cycle and in early pregnancy. The object of this paper is to outline the method currently used for the determination of plasma progesterone in our laboratory. Special attention is paid to the reliability at the lower limits of sensitivity of the method and to plasma progesterone levels during pregnancy. MATERIALSAND

METHODS

All organic solvents used were Analar Reagents from British Drug Houses, Ltd. Methanol and ethanol were purified by refluxing with NaOH and zinc powder and subsequent vacuum distillation. Acetic anhydride and pyridine were purified by vacuum distillation and stored in a desiccator. All small glassware (tubes, funnels, capillary funnels) was stored in concentrated sulphuric acid and shortly before use cleaned by rinsing with tap water (IO times) and distilled water (IO times). * Head of Department:Prof.Dr. W. P. PLATE. clin.

Chim. A&z, 8 (1963) 943-953

H. J. VAN DER MOLEN

944

Commercial steroid preparations were purified to constant melting point by recrystallization from organic solvents. Standard amounts of steroids were applied to paper chromatograms or added to solutions dissolved in methanol (containing IOO ,ug/ml), using an Agla micrometer burette (accuracy 0.0002 ml). Evaporation of large volumes was done using a rotating vacuum evaporator. Chromatography paper was, after cutting and marking the strips, continuously washed in a Soxhlet extractor with 80% methanol for at least 72 h. Immediately before use the strips were taken from the extractor and dried at 80” for IO min. All paper chromatograms were run in a room kept at 25”. Location of U.V. absorbing steroids on paper chromatograms was done by making a contact photo with Dalco daylight rapid copy paper, illuminating for 2-3 set with a Philips TUV 15 Watt lamp at a distance of 50 cm. Using the automatic developing apparatus for this paper, we obtained a contact photo in less than 30 set avoiding the use of a dark room. With this method it was possible to locate amounts as low as 0.5-1.0 ,ug of chromatographed progesterone. U.V. spectra were determined with a Zeiss-Spectrophotometer fitted with an M4Q monochromator, using microcells of 0.2 ml capacity. EXPERIMENTAL The quantitative

determination

of pure progesterone

OERTEL et a1.4 in their method

quantitated

the isolated

progesterone

by heating

at 60” for 15 min with an 80% ethanol-concentrated sulphuric acid 2: I (v/v) mixture. They determined the extinctions at 260, 290 and 320 m,u and used the modified ALLEN~ correction as given by BROWNS. Proof of the greater sensitivity of this reaction compared with the often used U.V. absorption of progesterone solutions, seemed to be their report of plasma progesterone concentrations in the normal menstrual cycle. However, comparing the ethanol-sulphuric acid reaction and the U.V. absorption in vacuum-distilled ethanol for pure progesterone, we came to the same conclusion as SHORT*, that there is hardly any difference between these two methods when the Allen correction is used. The spectra and calibration curves for pure progesterone are given in Fig. I and Table I (T = 0.50). With micro cuvettes (capacity 0.2 ml) it is possible with both methods to make a reliable determination of 0.1-0.2 pg of pure progesterone. For the U.V. determination the 95% reliability limits are approximately: Ecorr. & 2 x 0.005 = &rr. i 0.010; thus theoretically it is possible to differentiate from zero, an E,,rr. > 0.010, corresponding to a minimal concentration of 0.06 ,ug/o.z ml. In practice, however, several factors, such as contaminating impurities and the need for more than 0.2 ml for filling the cuvettes, decrease this sensitivity. We have also tried to use the highly sensitive reaction of progesterone with thiosemicarbazide as proposed by PEARLMAN AND CERCEO'. We had the same experience as SOMMERVILLE AND DESHPANDE~ in that despite the use of SCrUpUlOUSly cleaned glassware and extremely purified solvents, we were unable to standardize this reaction with pure progesterone for quantitative use, owing to high unreproducible blanks. Because of these results, and also because of the disadvantage that the sulphuric Clin. Chits.

Acta, 8 (1963)943-953

PLASMA

acid reaction eluates than

PROGESTERONE

DURING

PREGNANCY

945

destroys the progesterone and gives worse backgrounds with paper U.V. measurement, we used the U.V. absorption curves in all our

plasma progesterone

determinations.

The quantitative determination

of progesterone in eluates from paper chromatograms

As seen above, pure progesterone is easily measured by spectrophotometry; however, the elution after paper chromatographic isolation, also elutes impurities E

progesterone

4 pq/mi

:m

0.3

ethanol

- H,504

ethanol

300

Wavelength

(my)

o.3

Progesterone Fig.

I. Calibration

TABLE u.v.

CALIBRATION

concentration

T =

(pglml)

of determinations

0.30 Ecorr.l~glml S.D. T = 0.40 &orr./pg/ml S.D. T = 0.50 &orr.l,uglml S.D. .

I

CURVES

E COIT. = E 240 -

number

pg/ml

curves of pure progesterone.

OF

PURE

T.(&o

PROGESTERONE

+ E,,o) mean

0.5 26

1.0

2.0

40

47

3.0 31

4.0 I2

156

s.d.m.

0.044 0.008 0.038 0.007 0.034 0.008

0.043 0.007 0.038 0.006 0.034 0.006

0.042 0.005 0.038 0.005 0.033 0.005

0.043 0.006 0.038 0.004 0.034 0.005

0.041 0.003 0.038 0.003 0.034 0.004

0.043 0.006 0.038 0.005 0.034 0.005

0.00048 0.00042 0.00045

that interfere in the determination. To compensate for these impurities, it is common practice to use washed papers and to measure the progesterone-containing eluate against a corresponding paper blank eluate. To correct for interfering absorptions which are not corrected for by measuring against the blank, the correction as described by ALLEN~ is often applied. By this procedure, with washed papers measuring against a blank and using the Allen correction, we had difficulties in obtaining reproducible recoveries (see Table II). After chromatography of 0.5 pug of pure progesterone, the mean of 8 determinations gave a recovery of 0.68 pg with a standard deviation of 0.72 pg. C&t. Chim. Acta,

8 (1963) 943-953

s

E

?

2 :

54%

(in % of added)

I

F =

P > 0.25

1.66

‘44%

0.72

135%

0.68

3

3 us. I F = 8.07 P < 0.05

2 vs.

0.65

57%

0.28

7’7%

II

2

F = 3.58

1.04

3

0.99

47%

0.47

104%

~.

P < 0.01

P < 0.01

I .03

49%

0.49

99%

0.99

F = 3.82

0.53

25%

0.25

101%

I.01

I

I.0 ‘7

.-

0.99

24%

0.48

86%

1.72

2

1.20

29%

0.58

97%

* .94

3

F = 2.68

F =

I.05

77%

0.5’

84%

2.52

I 2

3.0 23

1.02

1.85

30%

0.90

83%

2.49

3

P <

0.01

P ;> 0.25

T.05

17%

0.51

79%

2.37

P ( 0.02 F = 3.13

F = 1.S4 P > 0.05

0.74

18%

0.36

90%

1.80

I

22

2.0

CHROMATOGRAPHY

The progesterone value is calculated from the CJ.V. absorption data with: (I) The T-value calculated from the blank of the same strip, measured against pure ethanol. (2) The mean T-value (0.36). (3) The ALLEN correction fT = 0.50).

(F-test)

Comparison of S.D.

0.62

0.27

95% significance limits (pg)

96%

2

0.5 8

0.58

1.12

__

0.48

I

S.D. recovery (pg)

(,ug)

with

(in o/0 of added)

Recovery

Calculation

Progesterone added (,ug) Number of determinations

TABLE

RECOVERYOF PROGESTERONE ADDED BEFORE

1.31

15%

0.60

3.06

35%

I.40

96%

3.84

2.96 74%

3

2

F = 9.88

P < 0.01

F = 1.78 P > 0.10

0.96

11%

0.44

90%

3.60

I

I2

4.0

0.97

33%

0.49

88Yb

2

1.67

56%

0.85

98%

3

F =

5.45

P <

0.01

F = 1.90 P < 0.01

0.78

24%

0.39

91%

I

mean 82

F

3

z

$

u

PLASMA PROGESTERONE DURING PREGNANCY

947

When we measured paper blank eluates against each other, it appeared that the principle underlying the Allen correction, i.e. the necessity of a linear background, is not valid. When the Allen correction is valid, we expect :

E 240- A(,??,,, + Eaao) = o and A = 0.5 (E being the measured extinction at the assigned wavelength). We calculated the factor A as: A L= ~~**~(E~~~ + E,,,) and found a value between 0.33 and 0.56 (mean 0.41; S.D. 0.08; n = 84). ZANDER AND VON MUNSTERMANN~ found that under their conditions the background absorption between 225 and 255 m,u was linear; this was substantiated by the results of DICZFALUSY lo with countercurrent distribution. In our case the value of A for the differences between paper blank absorptions from 225 to 255 rnp was 0.35-0.55 (mean 0.43; S.D. 0.07; n = 84). Though it is possible that a combination of impurities from paper and plasma might give a linear difference curve, thus justifying the Allen correction, we preferred the method of measuring all the samples and paper blanks dissolved in ethanol against pure ethanol. Thus the risk that an occasional high blank would not allow the measurement of a difference curve was avoided. For the blank we calculated the factor T: Es,, -

and calculated

T.(&,o

+

ho)

=

0:

T

=

&,o1(&m

+

.%,,I

for the samples:

E corr. = Em -

T. Wm + Lw).

The E,,,,, is compared with that of pure progesterone calculated with the same T. The mean value for T for ~atrn~ No. 20 paper, with extinctions at 220,240 and 260 rnp, was 0.36 (S.D. 0.05; n = 102). When extinctions at 225,240 and 255 m,u were used, the mean T value was 0.37 (S.D. 0.06; n = III). We consider the results in Table II as proof of this being an acceptable method of calculating the results under our conditions. Using the T-values we measured against pure ethanol; using the Allen correction we measured against a corresponding blank. Comparison of the standard deviations by the F-test shows that recoveries after paper chromatography are more precise using T-values than using the Allen correction. In each determination we prefer to use the T-value of the corresponding blank instead of using a mean value. The ‘~s~~n~ur~ ~~~~0~’ for the de~e~~~n~t~~n of $lasma

~~~~es~e7~~e

After comparing several steps from the methods as published by SHORT~Tllp 12, OERTEL et aL4 and ZANDER AND SIMMERIS, we use at present the following method, which contains most of the steps used by SHORT. Extraction. After addition of NaOH (final concentration 0.5%) progesterone is extracted from fresh heparinized plasma with 6 x 4 vol. of ethyl ether. The combined ether extracts are washed with 2 x 0.1 vol. of distilled water and evaporated to dryness. The residue is transferred with 3 x IO ml portions of petroleum ether (b.p. 40-60”) to a separatory funnel and extracted with 6 x IO ml portions of 70% methanol (the flask that contained the residue is washed with each IO ml aliquot of 70% methanol). The combined 70% methanol fractions are evaporated to dryness. C&n.Chini.

A&,

8 (rgh_x)943-953

948

H. J. VAN DER MOLEN

Chromatography. The residue is applied to a 3.5 x II cm “concentration strip” of washed Whatman No. I paper (see OERTEL I4 for details). Along with each plasma extract is taken a standard, applying I, 2,3 or 4 ,ug of progesterone on a concentration strip. Desoxycorticosterone (5 pg) is added to all concentration strips as a chromatography reference. The washed paper chromatograms used for the isolation of progesterone, are IO cm wide strips of Whatman No. I or 20 paper. On each strip is run one plasma sample and one standard at 2 cm from each side. Descending paper chromatography is performed in the Bush A system (petroleum ether (b.p. 8o-roe”)-methanol-water, IOO: 85 : 15). Equilibration is done overnight and development requires approximately 5-5.5 h for Whatman No. 20 paper and 2-2.5 h for No. I paper. The relative mobility of progesterone compared to that of desoxycorticosterone (RD~~) in this system, is between 4.7 and 6.0 (mean 5.2; n = 83) for Whatman No. 20 paper and between 3.7 and 4.6 (mean 4.3; n = 60) for No. I paper. After drying the chromatograms for a few El&ion of paper chromatograms. minutes in the fume cupboard, the steroids are located by means of a U.V. contact photo. The progesterone spot is usually 2 cm long and I cm broad. Whenever the progesterone spot is invisible, location is made through comparison with dcsoxycorticosterone and the standard. From the chromatogram 6 cm x 3 cm pieces, containing the progesterone spot, are cut. A blank is cut from the middle of the chromatogram at the same point between the standard and the plasma sample. The strips are eluted with 4 x z ml of vacuum-distilled methanol, using capillary funnels as described by BROICH’~. The eluates are evaporated in vacua. Quantitative determination. The residue is dissolved in exactly I ml of vacuumdistilled ethanol. Using micro-cuvettes, the extinctions are measured at 220, 225, 240, 255 and 260 m,u against pure ethanol. No result is calculated unless the curve obtained after subtraction of the paper blank from the samples, shows a peak at 240 mp. The progesterone content of the samples is calculated using the T-values and Ecorr. as described above. The “extended method” In some cases we extended the described method in the following way. After the U.V. spectrum has been measured the solution is quantitatively collected and evaporated in vacua. To the residue is added 0.5 ml of an acetic anhydride-pyridine, I: I (v/v) mixture. After being placed in a boiling water bath for I h, the solution is evaporated in vacua. The residue is concentrated in the same way as described in the standard method and rechromatographed in a petroleum ether (b.p. 8o-roe”)-acetic acidwater (100: go: IO) system. In this case, IO cm wide strips of washed Whatman NO. 2 paper are used. Equilibration is done overnight and development for 7-8 h (RDOC ranges from 4.8 to 5.7; mean 5.2; n = 38). Location, elution and quantitative determination of progesterone are performed in the same way as in the standard method.

PLASMA

PROGESTERONE

DURING

PREGNANCY

949

RESULTS

Reliability

qf the

method

To test the accuracy of the standard method we determined recoveries of progesterone added to plasma. Pooled pregnancy plasma (500 ml, stored in frozen state) was unfrozen and filtered until clear. To 20 ml fractions of this plasma were added different amounts (I, 2, 3 or 4 pug) of pure progesterone. Table III shows the results, the recoveries being determined by subtracting from each individual determination the mean progesterone value estimated from the 4 blank determinations (1.G pg). The mean recovery in this concentration range is 87% with a standard deviation TABLE RECOVERY

Progesterone added (pg) number of determinations Progesterone

found (pg)

Mean Recovery (pg) (in % of added) S.D. recovery (pg) (in % of added)

OF

PROGESTERONE

III ADDED

TO

PLASM.4

0

2

3

4

4

7

5

4

3.10 4.20 4.10 3.80

4.85 4.14 5.05 5.45

3.92 2.32 77% 0.45 15%

4.87 3.27 820/6

1.20 1.60

2.60 2.20

I .9o 1.70

2.40 2.45 2.50

4.00 3.10 2.70 3.30 3.40 3.15 3.50

1.60

2.43 0.83 83% 0.13 13%

3.31 I.71 85% 0.37 I8 %

0.30

0.50 13 %

mean

87% 0.37 16%

of 16%. When this value is compared with the recovery from the chromatography in Table II, there seems to be a small loss of progesterone in the steps preceding chromatography. The results in Table III also give an impression about the precision of the method. For the different concentrations, including the plasma blank, the standard deviation ranges from 0.13-0.50 pug, with an overall mean of 0.37 ,ug. Comparing these values with those of Table II, they appear to be of the same order, indicating that the precision is not influenced by possible impurities from the plasma. With regard to the sensitivity of the method as a whole, it can be concluded from the precision that, accepting 95% confidence limits, approximately 0.72 ,ug can be distinguished from zero. This value again is in agreement with the results in Table II. SpeciJicity of other methods for the determination of plasma progesterone has been claimed on several grounds (I.R.-spectrometry, countercurrent distribution). BUSH’~ proposed, mainly on theoretical grounds, that plasma progesterone should be purified not only by one paper chromatographic separation of the free steroids, but also by additional chromatography after acetylation. Steroids that might occur in the same chromatographic region as progesterone are .@-pregnan-3/3-ol-zo-one and the androstane-3, r7-diones. If these steroids occur in the progesterone fraction in our method, we would not except too much interference as they do not absorb U.V. radiation. Moreover, we did not succeed in demonstrating (for other purposes) 5/L Clin. Chim. Acta, 8 (x963) 943-953

H. J. VAN

950

DER MOLEN

TABLE RECOVERY

OF PROGESTERONE

ADDED

Recovery

Pro-

rst

gesterone

added

I

I

_-_0, /o

0.73 1.30 7.20 1.60

2 2

3.00 2.30 2.60 2.40 3.10 2.80

137% 100%

I.20

100%

.30

1.50 I .go

50% 00 %

2.10

91% 111%

2.90 2.00 2.20

83% 71%

z-30

82% 96% 26q/,

TABLE ISOLATED

FROM

pg isolated after 1st chromatography

PLASMA

USING

0.45

I.10

0.91

1.30

0.95 1.40

2.00

I.50

2.30 2.40

2.50 1.62

3-50 5.20

3.03 4.00 5.66 6.40

5.90 7.70 12.01

V REPEATED

,ag isoEated after chromatogra$hy

2nd

0.34

I .45

of I

8 1% I joy’,

73% 11%

PROGESTERONE

“/b

1.30

70%

Mean S.D.

2 in

4.10

70% 77% 65% 60 “/:, 78V

CHROMATOGRAPHY

1.00

I

100%

2.10

PA’ER

0’ /o

fa

73% 65% 60% 80% 83%

2.50

3 3 3 3 1 4 4 4

REPEATED

Recovery after 2nd chromatography 2

after

Pug

2

IV USING

chromatography

.________-.

cwi

TO WATER

PAPER

2 in

yo

CHROMATOGRAPHY

of I

132% 82% 73% ;$ 0 109% 67% 86” 772 96% 83% 92%

11.00

&lean S.D.

89% 17%

pregnan-$?-ol-zo-one in up to I 1 of pregnancy plasma with a method sensitive enough to detect less than IO pg. For several samples, we compared the yields of progesterone using both the standard method and the extended method. Table IV shows the comparative results for pure progesterone added to water; Table V gives the results by both methods for several unknown plasma samples. Comparing the percentages of progesterone isolated after the second chromatogram relative to those after the first chromatogram, there is no significant decrease for the plasma samples compared with the recoveries of pure progesterone (d = 1.1; n = 22; P >o.Io). There is thus no reason to conclude that additional purification of progesterone is achieved in the extended method. Clin. Chim. Acta, 8 (1963) 943-953

PLASMA

The combined of this standard

Plasma

results

method

PROGESTERONE

DURING

on the reliability

PREGNANCY

of the method

for the determination

9s

appear to justify

the use

of plasma progesterone.

firogesterone concentrations

All results reported in this paragraph were obtained by the standard method. The results were not corrected for losses during the determination. In pooled plasma (8 samples measuring up to 50 ml) from normal women we were not able to demonstrate progesterone. From 6 different subjects, we obtained blood from the maternal part of the cord,

x

I

*

a’ 0

‘:

0

* a ~“~*r*~*~ IO

l

*

xx x

lxjx-

**

xx

20

Weeks

a *

after

30 last

8

a

2

c 8

40

menstrual

’

period

Fig. 2. Plasma levels of progesterone in peripheral venous blood during pregnancy of 62 women (X singles; 0 twins) portus abortion

0’

10 Weeks

20 after

lost

30 menstrual

40 period

Fig. 3. Plasma levels of progesterone in peripheral venous blood at various times during pregnancy

immediately

after it had been cut. The progesterone concentrations found were: 15.5, 28.4 and 78.8 ,ug per IOO ml plasma. During pregnancy we collected from several women peripheral blood in heparinized tubes, all samples giving approximately 20 ml of plasma. Fig. 2 shows the result of plasma progesterone determinations in peripheral venous blood from 62 different women during normal pregnancies, resulting in the deliveries of normal babies. Fig. 3 shown the results for women where it was possible to collect more than one sample during pregnancy. 81.0, 55.0, 37.0,

Clin.

Chim.

Ada,

8 (1963)

g.+j-g53

H. J. VAN DER MOLEN

952

DISCUSSION Though

we can determine

with reasonable

accuracy,

the final assay using U.V. hope,

0.5-x.0

absorption

that the ethanol-sulphuric

amounts,

pg of progesterone is the weakest

acid reaction

likely,

plasma

link in the method.

might

method

paper impurities, Our first

allow us to determine

smaller

was not confirmed.

Unless it is possible to collect relatively highly

by the standard

we feel that mainly due to contaminating

that for the accurate

of normal

derivative

method

males

and females

as developed

large plasma

estimation

samples,

of small amounts

or during

early

it seems therefore of progesterone

pregnancy,

a double

in

isotope

by WOOLEVER AND GOLDFIEN~’ must be the method

of choice. The fact that we did not find progesterone subjects,

assuming

progesterone

a sensitivity

levels in normal

ml. This assumption

in 50 ml plasma samples from normal

of our method subjects

is validated

of at least I ,ug, indicates

might

be expected

that plasma

to be less than 2 pg/Ioo

by results of WOOLEVER 4ND GOLDFIEN~‘, but dis-

agrees with the results of OERTEL et al.4 and HERVB AND SERGENT~~, who found values up to 5 pg/Ioo

ml.

We found levels in cord blood in the same range as those reported by RUNNEBAUM AND ZANDER~~ and ZANDER~~ before cutting

for blood collected

by puncturing

the cord. This could be expected

the Vena Umbilicalis

as we only collected

blood

coming

from the placenta. In peripheral plasma during pregnancy to quantitative

we would not attach too much importance

values lower than 5 ,ug/Ioo

with sufficient reliability, The values

in Figs.

ml (assuming

where our samples

2 and 3, are in better

who also found scattered

values throughout

agreement

pregnancy,

who showed a steady rise with progress of pregnancy. more than one sample progesterone

for the same subject,

concentration

simply

on grounds

absolute

amounts

twin pregnancies

variation

of bad recoveries.

Again,

Where

values

it was possible to collect increase in the plasma

is seen (Fig. 3). However,

in

in plasma levels that can not be explained considerable

from case to case. We found, the estimated

with those of ZANDER’~,

than with those of SHORP,

a more distinct

with progress of pregnancy

some cases there is a considerable

that I ,ug may be estimated

are in the order of 20 ml).

differences

in agreement

are often,

are found

in the

with SHORTY, that for

but not invariably,

in the higher

ending in abortion,

did not show

range. Our few observations

concerning

pregnancies

any marked decrease or very low levels of the progesterone

concentration

in peripheral

plasma. ACKNOWLEDGEMENT The author expresses criticism

his gratitude

during preparation

to Dr. A. C. BROWNIE for helpful advice and

of the manuscript. REFERENCES

1 IV. H. PEARLMAN, in: H. N. ANTONIADES (Ed.), Hormows Company, Boston, 1960, p. 415. 2 R. V. SHORT, in: C. H. GRAY AND A. L. BACHARACH Press, New York and London, 1961, p. 379.

in humna?z plasma, (Eds.),

Hormows

Little, Brown and in blood,

Academic

C/in. (‘hitn. Actu, 8 (1963) 943-953

PLASMA

PROGESTERONE

DURING

953

PREGNANCY

3 J. ZANDER, in: R. I. DORFMAN (Ed.), Methods in hormone research, Vol. I, Academic Press, New York and London, 1962, p. gr. 4 G. W. OERTEL, S. P. WEISS AND K. B. EIK-NES, J. Clin. Endocrinol., rg (1959) 213. 5 W. M. ALLEN, J. Clin. Endocrinol., IO (1950) 71. B J. B. BROWN, Biochem. J., 60 (1955) 185. 7 W. H. PEARLMAN AND E. CERCEO, J. Biol. Chem., 203 (1953) 127. 8 I. F. SOMMERVILLE AND G. N. DESHPANDE, J. CZin. Endocrinol., 18 (1958) 1223. Q J. ZANDER AND A. M. VON MUNSTERMANN, Klin. Wochschr., 34 (1956) 944. lo E. DICZFALUSY, Acta endocrinol., 20 (1955) 216. ii R. V. SHORT, J. Endocrinol.. 16 (1958) 415. i2 R. V. SHORT, Mem. Sot. Endocvinol., 8 (1960) 86. 13 J. ZANDER AND H. SIMMER, Klin. Wochschr., 32 (1954) 529. I4 G. W. OERTEL, Naturwiss., 43 (1956) 17. i5 J. R. BROICH, J. Chromatog., 5 (1961) 365. 16 I. E. BUSH, The chromatogra+hy of steroids, Pergamon, Oxford, 1961, p. 316. 17 C. A. WOOLEVER AND A. GOLDFIEN, in: Abstracts of papers presented on the international congress on hormonal steroids, Milan, Excerpta Medica, International Congress Series, No. 52, Amsterdam, 1962, p. 81. 18 R. HERV~ AND P. SERGENT, Gyne’col. et ObstLt., 60 (1961) 55. 10 J. ZANDER, in: C. W. LLOYD (Ed.), Recent progress in the endocrinology of reproduction, Academic Press, New York and London, 1955, p. 255. so B. RUNNEBAUM AND J. ZANDER, Klin. Wochschr., 40 (1962) 453. z1 J, ZANDER, in: Progesterone and the defense mechanism of pregnancy, CIBA Found. Study Group g, J. & A. Churchill, London, 1961. p. 32. Cl&. Chim. Acta, 8 (1963) 943-953

APPLICATION REACTION

OF A FERROCYANIDE-PHOSPHOMOLYBDATE

TO

AN

AUTOMATED

DETERMINATION

OF

SERUM

GLUCOSE B. FINGERHUT,

R.

FERZOLA

AND

W.

H.

MARSH

Institute of Pathology, Kings County Hospital Center and Department of Pathology, State University of New York, Medical Center, Brooklyn, N. Y. (U.S.A.) (Received

January

Iqth, 1963)

SUMMARY

An automated method for the determination of serum glucose is presented. It involves the reduction of alkaline ferricyanide to ferrocyanide by glucose and the subsequent reaction of ferrocyanide with molybdate in phosphoric acid medium to form a colored product. At the rate of 40 determinations per hour the method requires the use of 0.2 ml serum, and can determine glucose levels within the range 0-500 mg%. Recovery experiments gave yields of 98.8-100.5 mg%. The standard deviation for 50 specimens analyzed in duplicate was 3.51 mg%. Results obtained by this method average 4.4 rng% less than the Folin-Wu values, and 3.5 rng% less than the conventional automated alkaline ferricyanide reduction procedure.

INTRODUCTION

Automated blood glucose analysis as performed by the instrumental system, the Auto Analyze+, has become more frequently used in clinical chemistry laboratories Cl&. Chim. Acta, 8 (1963) 953-959