Improved estriol determination in a continuous flow system

BIOCHEMICAL

MEDICINE

Improved

8,

188-198

Estriol

(1973)

Determination

in a Continuous

Flow System M. LEVER,’ Pathology

J. C. POWELL, Department, Auckland,

New

AND

Greenlane Zealand

S. M. PEACE Hospital,

Received October 18, 1972; revised January 8, 1973 Urinary estriol determinations are widely used as an index of fetalplacental function (1, 2). When large numbers of these determinations are requested the assay systems of choice are usually based on the Kober color reaction (3) or the Ittrich fluorescence reaction (4). Automated procedures have been described (5-S) which enable up to 150 samples per day to be assayed, but a greater capacity is expected to be required in this laboratory. The procedure described here is based on a modification of the Ittrich fluorescence reaction. The urine is extensively diluted to reduce interferences. All solvent extraction steps are eliminated, which makes it possible to sample specimens at rates up to 60/hr. Also described is a rapid manual version of the procedure. This ensures continuity of comparable results in case of breakdown. Also single, urgent specimens can be assayed in less than 30 min without operating the automated system. METHODS Instrumentation The automated continuous flow system was developed with Technicon Basic AutoAnalyzer modules except for the fluorometer. This was a Photovolt filter fluorometer (model 520-M) adapted by fitting it with a 100-W tungsten filament lamp as light source, 530 nm (excitation), and 560 nm (emission) interference filters (Baird-Atomic) and with a simple glass tube as a flow cell. Polyethylene tubing was used wherever possible. Other fluorescence measurements were made with an Aminco-Bowman spectrophotofluorometer fitted with a xenon arc lamp and an RI36 photomultiplier. Mirrors and IO-mm slits were placed in the cell housing. *Correspondence to Dr. M. Lever, Pathology Department, Auckland 3, New Zealand. Copyright All rights

188 @ 1973 by Academic Press, Inc. of reproduction in any form reserved.

Greenlane

Hospital,

ESTRIOL

DETERMINATION

189

Gas chromatography of estrogens was carried out with a HewlittPackard model 402 instrument fitted with a flame ionization detector. Reagents Standards. Estriol obtained from Steraloids, Inc. (Pawling, NY) and from Sigma Chemical Co. (St. Louis, MO) gave identical responses in the assay systems studied. A stock solution of estriol was made by dissolving 144 mg estriol in 10 ml cold, concentrated sulfuric acid and allowing this to stand at room temperature for 20-60 min. The resulting solution (containing estriol sulfonates and sulfates) was then poured into ice-cold water and made up to 1 liter with further water. If desired, creatinine can be dissolved in this to make a combined estriol-creatinine standard. The stock standard (equivalent to 500 pmoles/liter estriol) is diluted with 0.1 M H,SO, to make the working AutoAnalyzer standards. These are quite stable in aqueous solution and can be stored in polythene bottles without loss. These solutions are suitable also for the manual procedure, and were calibrated by comparing them by the manual procedure with standard solutions of estriol in ethanol In the calorimetric and gas chromatographic procedures, standard solutions of estriol in ethanol were put through the final stages of the assays but not through the initial hydrolysis and extraction steps. Kober Reagents. (1) AutoAnalyzer. Quinol (3.2%, w/v) was dissolved in cold, concentrated sulfuric acid, and allowed to stand 30 min or longer to ensure complete sulfonation. The quinol solution (3 vol) was then diluted with 1 vol aqueous ferrous sulfate solution ( 2.4 g FeSOJ 100 ml), with cooling. This reagent is then ready for use. It is stable for several days if protected from excessive exposure to air. (2) Manual procedure. Quinol in coned sulfuric acid (3 vol) is diluted with 1 vol aqueous ferrous suIfate sohrtion and 1 voI T-I,0 to give a reagent approximately the same as that which enters the oil bath in the AutoAnalyzer procedure. Fluorescence Reagent f AutoAnalyzer). The fluorescence reagent consists of 500 g each of trichloroacetic acid and chloral hydrate, with distilled water to make up 2 liters of solution. Procedures AutoAnalyzer Procedure. The continuous flow system (Fig. I) involves ( 1) 4O-fold dilution of urine. (2) A portion of this is resampled without debubbling (further portions can be resampled for simultaneous creatinine and other determinations) and diluted approximately y-fold with segmented Kober reagent. (3) The diIuted Kober reagent (approx. 64%

190

LEVER,

Po~EL~+A~

PEACE

SAMPLER II LO- 2/l

WATER JACKETED SINGLE MIXER TUNGSTEN LIGHT SOURCE EXCITATION 530 nm EMISSION 560 m-n l ACIOFLEX

tOOW

FLUOROMETER

FIG. 1. Flow diagram of the continuous flow estriol procedure. Polyethylene connecting tubing (1.2 mm id) used in all lines containing acid. Pump tube internal diameters given in inches.

v/v H,SO, with 2% quinol and 0.5% ferrous sulfate) is heated for about 12 min at 129°C. (4) It is mixed with about 4.5 vol of fluorescence reagent and cooled. There is no extraction step at this stage. The fluorescence in this solution is measured with excitation wavelength 530 nm and emission wavelength 560 nm. Short Manuul Procedure. The chemistry of the manual procedure is similar. Urine is prediluted lo-fold. Diluted urine ( 10 ~1) is added to 1.5 ml Kober reagent in a Brown fluorometer cuvette (9) and mixed on a vortex mixer ( 10 set). The mixture is heated in a polyethylene glycol bath (fume cupboard) at 120% for 20 min and then cooled on ice. A layer of chloroform (0.7 ml) is added on top of the acid, then 1.5 ml 10% trichloroacetic acid in water. The Brown cuvettes are stoppered, shaken vigorously for 60 set on a rapid vertical shaker (lo), and then centrifuged. The fluorescence in the chloroform phase is read with excitation 530 nm and emission 550 nm. Calorimetric Procedure. The calorimetric method of Brown et al. ( 11) was carried out by technical staff in an adjacent routine laboratory. Gas Chromatographic Procedure. Hydrolysis and extraction steps were carried out according to method No. 2 of Brown and CoyIe (12). The estrogens were methylated with dimethylsulfate and gas chromatography of the ethers was carried out with a primed column of 3% SE 52 on SO100 mesh Diatoport S in a 100 X 0.4-cm column at 230°C. The carrier gas was N, (35 cm3/min) and cholestane the internal standard (method of Dr. J. T. France, personal communication).

ESTRIOL

191

DETERMINATION

Evaluation Urines on which estriol determinations had been made colorirnetrically were assayed by the AutoAnalyzer procedure. When Iarge discrepancies between the results by the two methods were observed, the urine was also assayed by the gas chromatographic procedure. Urines from males and nonpregnant females were also assayed with and without different amounts of estriol added either in ethanol or as sulfonated derivatives. RESULTS

Kober Reagent Higher estriol estimates are obtained when the urine is diluted before a direct estriol determination, and our early trials confirmed the claim of Beischer and Brown (1) that occasional urines give spuriously low results even with a procedure that usually gives greater than 90% recovery. Two urine specimens from nonpregnant patients were observed to have this effect on the fluorescence of added estriol (Fig. 2A), and this could be greatly reduced by adding ferrous suIfate to the Kober reagent (Fig. 2B). Replacing quinol entirely with ferrous sulfate gave 50% of the fluorescence yield; a lower response was also observed by Brown (13). Also, with ferrous sulfate alone in the Kober reagent, strong quenching was observed when urine was added. Increasing the quinol concentration S-fold produced only a marginal improvement. Ferrous sulfate and quinol together seem to be most effective, and this combination also eliminated interference from traces of peroxides in solvents.

W Y 52 20!L

cl

lo-Fe WI&

k!

',

'.

s

iii

-.

--a-

0

ypl----

0

10

20

KOBER REAGENT

(ml)

FIG. 2. Effect of ferrous sulfate on Kober reaction interferences. Estriol standards (50 pmoles/liter ) added to nonpregnancy urines ( N and W) and to water; 10 pl solution added to Kober reagent. After heating, 1.5 ml Kober reagent transferred to Brown cuvette. (A) Kober reagent without FeSOa (B) Kober reagent with 0.5% FeSO+.

192

LEVER,

POWELL,

AND

PEACE

Standurds Using “Sigma” estriol, 10 replicate sulfonated standards were prepared by dissolving 10 mg estriol in 1 ml concentrated sulfurnic acid. After standing these were each diluted to 100 ml with water and the resultant solutions were assayed for estriol. By the manual procedure the coeficient of variation was 8% and by the automated procedure 2.2%. Replicate assays on the same standard had a coefficient of variation of 5% (n = 16) by the manual procedure and 0.7% (n = 20) by the automated procedure. Standards of estrone, estradiol, and the 3-methyl ethers were also prepared by the sulfonation procedure. In the automated assay estrone gave 66%, estradiol 8%, estriol &methyl ether 79%, and estrone n-methyl ether 64% of the response of estriol ( all on a molar basis). When the native estrogens were sampled (in 25% ethanol) only estriol gave a response closely similar to its sulfonated derivative (Table 1). Estriol 3-methyl-

Relative

Epiestriol Ethynylestradiol Me&ran01 Testosterone Progesterone Pregnanediol Pregnanetriol 17a-hydroxyprogesterone 1 lp-hydloxyprogesterone Cortisone Cortisol Corticosterone 11 Deoxycorticosterone 11 Deoxy l’lcu-hydroxycorticosterone 21 Deoxycortisol Androsterone Dehydroisoandrosterone

ll,%hydroxyandrosterone Androstenedione d Steroid

solutions

(100

PM

(estriol

100 55 n‘7 100 4 5 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0

Estriol Estrone Estradiol

procedures.

resnorrse

in 25O./u ethanol)

analyzed

= 100)

100 4 1ti 100 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 by

manual

and

automated

ESTRIOL

193

DETERMINATION

ether gave 58% of the response, whereas estradiol and estrone gave low or zero results. This is believed to be because native estrogens are absorbed into plastic pump tubing. In the manual procedure, estriol standards in 25% ethanol and sdfonated estriol standards give almost the same results. Estrone standards in ethanol give 15%-20% higher responses than sulfonated standards, whereas estradiol seems to be substantially destroyed in the sulfonation process. Continuous

Flow

Kinetics

Figure 3 shows the kinetics of the continuous flow system, plotted as described by Walker et al. (14). From this, the lag-phase factor (a) was calculated to be 0.10 min and the exponential factor (b) 0.20 minutes. According to Walker et al. (14) the sampling time to obtain 95% peaking should, therefore, be 0.6-0.7 min, so that this procedure is potentially capable of operation at 60 samples/hr. With the present interim instrumentation it is being operated at 40 samples/hr, which is compatible with concurrent creatinine estimations. In a trial under these conditions, 250 ,& standard gave 91% steady state and the interaction between 250 & standards and 50 ,LM standards was less than :3%. These resuhs suggest that there was considerably more carry-over than expected from the continuous flow kinetics. This difference is at least in part instrumental, and is not constant. The observed carry-over is satisfactory with the present sampling rate of 4O/hr.

TIME (MINUTES) FIG. 3. Kinetics of continuous curve plotted as difference from

flow

plateau

system (A) fluorescence,

rise curve (B) after ( 14).

fall

curve.

Rise

194

LEVER,

POWELL,

AND

PEACE

When the reaction is carried out at 95’C, using 85% sulfuric acid in the Kober reagent, a was found to be increased to 0.27 min and b to 0.50 min. These results are compatible with a sampling rate of 30/hour. Specificity

The manual procedure is specific for the natural estrogens (Table 1)) and does not give a significant response with the commonly used synthetic hormones. Corticosteroids and progesterone metabolites do not cause significant interference, The specificity of the automated procedure would be expected to be similar, but the loss of free steroids makes it difficult to be certain that estradiol conjugates are being detected. When the reaction is carried out at 95°C much higher relative responses are obtained with e&one and estradiol (two to three times estriol on a molar basis). This can be corrected by raising the sulfuric acid content of the Kober reagent to 85%, which gives a sulfuric acid concentration in the heating coil of 74%. Under these conditions the specificity is similar to the results shown in Table 1. Evaluation

When 521 pregnancy urines were assayed by both calorimetric and automated procedures, a correlation coefficient of 0.90 k 0.01 (SE) was obtained. The regression equation (estriol concentration in @moles/liter), with calorimetric results y and automated result X, was: Alternatively,

y = 0.52x + 9 the regression can be given as x = 1.55y + 1.

Results obtained by the rapid manual procedure agree closely with the automated procedure. Urines with large deviations from the regression line were also assayed by the gas chromatographic procedure. The correlation coefficients of TABLE CORRELATION

Calorimetric Automated !dc

2

OF METHODV

Calorimetric

Automated

glc

-

0.69

0.69 0.73

-

0.73 0.96 -

a Specimens (n = 22) with poor agreement between procedures were also assayed by gas chromatography.

0.96 automated

and calorimetric

ESTFUOL

195

DETERMINATION

200

ESTf?lOLCONCENTRiVI~N(p rno$~O/Mel FIG. 4. Response of assays to different estriol concentrations (A) automated system: sulfonated estriol in 0.1 M H,SO* (B) manual system: estriol in 50% ethanol, diluted with water before sampling.

these by the three methods is shown in Table 2. The gas chromatographic procedure, although highly specific for estiol, involves the same hydrolysis and extraction steps as the calorimetric procedure, and similar losses could be expected in the two procedures. Therefore, the correlations in Table 2 suggest that the automated procedure is a more reliable guide to estriol levels than is the calorimetric method, and thus the first regression equation is the more useful. Figure 4 shows that both the automated and manual procedures give linear responses over the range of urinary estriol levels that are usually encountered. No cases were found in which standards added to urine gave a response different from standards added to water. Recoveries of TABLE EFFECT

OF ADDED

3

ALBUMIN AND GLUCOSE BY THE CONTINUOUS FLOW

ESTRIOL SYSTEM

ON

ESTIMATIONS

To Decrease in assay response Additive

concentl-ation (g/liter) 5 10 20 30 50 100

With albumina 0 0 0 0 3 5

With glucose* 0 4 15 22 41 63

a pooled urine from pregnant patients (estriol content 80 pmoles/l) eatfiol with various levels of (i bovine serum albumin and * glucose added.

assayed for

196

LEVER,

POWELL,

AND

PEACE

added sulfonated standards from 12 urine specimens (from males and from nonpregnant and pregnant females) ranged from 91-103X (average 98%). TabIe 3 shows the effect of albumin and glucose on the automated procedure. Albumin is unlikely to interfere with estriol determination. In rare extreme cases glucose could significantly decrease estriol estimates. This problem can be overcome if prediluted specimens are also assayed when the clinical particulars suggest that very high glucose levels are possible. DISCUSSION

Previous automated estriol procedures (5-S) have included extraction steps, either to separate the steroid from the sample, or to separate the fluorescent derivative from the Kober reagent. As a consequence, the rate of flow of the unsegmented stream through the detector flow cell is low. This could be expected to lead to a low sampling rate (14), usually 20/hr or 30/hr. Furthermore, caution is needed to ensure reliable extraction of Kober derivatives into chlorinated hydrocarbons (10, 15), preferably with vigorous agitation. Under the conditions in a continuous flow system the extraction efficiency will be low and, therefore, likely to be dependent on other urine components which can affect partition coefficients. Extensive dilution of the urine reduces this problem, but in the method described here it is eliminated completely. Dilution also greatly reduces the so-called quenching effect of urine on estriol fluorescence. This effect is not true quenching since it occurs in the Kober reaction, and is probably caused by unknown oxidants. Quinol in the Kober reagent has been supposed to be effective as a reducing agent (13), but other more powerful reducing agents are less effective. The addition of quinol helps to reduce the adverse effect of urine, and this is greatly assisted by the addition of ferrous sulfate, although ferrous sulfate alone is not effective. The addition of ferrous sulfate also appears to have a small catalytic effect on the Kober reaction. These observations are consistent with a readily oxidized free radical being involved in the conversion of the labile intermediate termed XRx4, as described by Jones and Haenal (16). A problem with automated estriol procedures is the choice of standard. Free estrogens are not sufficiently soluble in water to allow their use in aqueous standards, and they partition readily into soft plastics such as are used in the Technicon AutoAnalyzer. Organic solvents such as ethanol have significant effects on the Kober reaction, and at 120°C they lead to sufhcient pressure in the heating coil to adversely affect the bubble pattern. The estriol derivatives formed in cold sulfuric acid (pre-

ESTRIOL

DETERMINATION

197

sumably sulfonic acids and sulfate esters) are readily water soluble and behave as estrio1 in the Kober reagent. The corresponding e&one derivatives are also usable, but estradiol appears to undergo further reaction even in cold sulfuric acid. PresumabIy estradio1 conjugates are determined by the automated procedure with 50-60% of the molar response of cstriol, but this has not been demonstrated. These uncertainties do not affect the reliability of this method for the determination of estrogen excretion in pregnancy, when estriol is greatly predominant. The choice of temperature depends upon the needs of a particular laboratory. The initial work in this laboratory was all carried out at 12O”C, and this has proved satisfactory. A 95°C heating bath is a standard Technicon AutoAnalyzer module. At this temperature a heating time of at least 30 min is required to reach maximum color development even with 75%sulfuric acid in the Kober reagent, and although substantial reaction occurs in 10-15 min the response is too dependent on precise timing to make this a viable manual procedure, In a continuous flow system this is not a serious problem, and the automated method for estriol can be carried out quite reliably at 95°C provided the sulfuric acid concentration in the Kober reagent is raised to 85%to eliminate high results from estradiol and estrone. However the viscosity of the Kober reagent in the heating coil is greater both because of the higher sulfuric acid concentration and because of the lower temperature, which means that less favorable flow characteristics are obtained and a slower sampling rate must be adopted. In order to make it possible for estriol estimations to be carried out at 60 samplesihr, both the Iag-phase factor (a) and the exponential factor (b) (14) are reduced by using polyethylene tubing vvhenever possible, resamphng after dilution is made without debubbling, and the debubbling is brought as close to the flow-cell as possible. The use of a single phase throughout decreases both factors, and the rapid flow-rate through the flow cell is especially advantageous. At present the system is operated at only 40 sampleslhr because the present fluorometer is old and has a relatively slow response and considerable noise, and because compatible creatinine and protein procedures are still being examined. When these problems are overcome it is expected that a combined instrument will be constructed to determine all three parameters at 60 samples/hr, a service which will be necessary in the near future. SUMMARY

A continuous flow system, without solvent extraction steps, is described capable of determining urine estriol at up to 60 samples/hr. Estri01 dissolved in coned H,SO, and then diluted with water provides a

198

LEVER,

POWELL,

AND

PEACE

stable and water-soluble standard, and ferrous sulfate is added to the Kober reagent to eliminate spurious low results. A compatible, rapid manual method is also described. The continuous flow system is shown to be a more reliable measure of urinary estriol than is a commonly used semiautomated calorimetric procedure. ACKNOWLEDGMENTS The gas chromatographic analysis could not have been carried out without the assistance of Dr. J. T. France, who provided facilities and advice for this work. The calorimetric assays were carried out by Mr. M. P. Grady, and his assistants. Dr. C. W. Small is thanked for helpful discussions. REFERENCES 1. BEISCHER, N. A., AND BROWN, 2. OAKEY, R. E., Vitam. Harm.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

J. B., O&et. Gynecol. Szrro. 27, 303 ( 1972). (New York) 28, 1 ( 1970). KOBER, S., Biochem. Z. 239, 209 ( 1931). ITTFXCH, G., Z. Phys. Chem. 312, 1 (1958). UA CONAILL, D., AND Mum, G. G., Ckn. Chem. 14, 1010 (1968). BARNARD, W. P., AND LOGAN, R. W., Clin. Chim. Acta 29, 401 ( 1970). CAMPBELL, D. G., AND GARDNER, G., C&n. Chim. Acta 32, 153 ( 1971). HAINSWORTH, I. R., AND HALL, P. E., Clin. Chim. Actu 35, 201 ( 1971). BROWN, J. B., MACNAUGHTAN, C., SMITH, M. A., AND SMYTH, B., J. Endocm’nol. 40, 175 ( 1968). POWELL, J. C., N.Z.J. Med. Lab. Technol. 26, 69 (1972). BROWN, J. B., MACLEOD, S. C., MACNAUGHTAN, C., SMITH, M. A., AND SMYTH, B., J. Endocrinol. 42, 5 ( 1968). BROWN, J. B., AND COYLE, M. G., J. Obstet. Gynecol. 70, 219 (1963). BROWN, J. B., J. Endocrinol. 8, 196 (1952). WALKER, W. H. C., PENNOCK, C. A., AND MCGOWAN, G. K., Clin. C&m. Acta 27, 421 (1970). BROWN, J. B., AND BEISCHER, N. A., Obstet. Gynecol. Sure. 27, 205 (1972). JONES, H. A., AND HAENEL, R., Nature (London) 215, 1381 (1967).