197
Clinica Chimica Acta, 60 (1975) 197-204 0 Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6931
ESTROGEN ANALYSIS CORRECTED
M. RYAN Nuffield
IN PREGNANCY
URINE:
AN ARTIFACT
and B.C. GRAY Department
(Received
October
of Clinical
Biochemistry,
Radcliffe
Infirmary,
Oxford
(U.K.)
10, 1974)
Summary The effect of glucose and certain other compounds in reducing the yield of estrogen in urine analysis has been shown to be due to the action of the aldehyde group during acid hydrolysis. A means of removing this interference with borohydride is given, and the nature of the reaction between glucose and estriol is discussed.
Introduction A frequent problem encountered in urinary estrogen assay is the incomplete recovery of estrogen following acid hydrolysis. Several workers have demonstrated a decrease in yield when estrogen conjugates have been hydrolysed in the presence of certain compounds, notably glucose [l-5], other saccharides [3,4] and some medicaments [5,6]. Although the method of enzymatic hydrolysis of the conjugates is not susceptible to this iqterference, that of acid hydrolysis remains the more common procedure. It has been shown that estrogen recovery can be increased by dilution of the urine 5-25-fold [1,2,5], or by separation of the estrogens from urine without prior hydrolysis by precipitation [7] . Recent automated methods [8,9] in which the urinary estrogens react directly with Kober reagent have also been found to give erroneously low results in the presence of these same compounds unless a large dilution step is included in the procedure. Similar problems have been experienced in the related field of urinary 17-hydroxycorticosteroid analysis when glucose was present in the urine. One solution [lo] was the addition of borohydride in excess of that required for the reduction of the 0x0 grouping, so that all the glucose present was reduced to sorbitol. It was considered that the decreased yield in the estrogen analysis might be due to the presence, or production during hydrolysis, of aldehydes, and that these aldehydes reacted with estriol to form a complex which is not
198
measured by either the Kober reaction or gas chromatography. Reduction to an alcohol with borohydride prior to hydrolysis would nullify their action. In this report the effect upon estrogen recovery of heating acidified solutions of estriol in the presence of glucose or certain other sugars, aldehydes, ketones, carboxylic acids and alcohols has been studied. The action of borohydride in removing the effect of decreased recovery has been demonstrated with most of these compounds.
Materials and Methods All 24 h specimens of urine were collected in bottles containing a small quantity of thiomersal (B.D.H. Chemicals, Ltd., Poole, England) as preservative. The volume was recorded and specimens of less than 2 1 were diluted to this volume. This had the two-fold advantage of making most urinary estrogen concentrations fall within the range of a standard calibration curve, and of simplifying the final calculations where urinary values were expressed in mg estrogen per 24 h. Urines were routinely tested for glucose with Clinistix (Ames Co., Stoke Poges, Slough, England).
Estrogen estimations (a) Automated
method. The Kober-Ittrich procedure of Campbell and Gardner [9] was modified to give the following method. Urine sampled at 0.16 ml/min was mixed with quinol (1.5 mg/lOO ml) in sulphuric acid (65 : 100, v/v) pumped at 1.6 ml/min. This mixture was heated at 120°C for 12 min, diluted with water, cooled and the Kober complex extracted into 2% w /” p-nitrophenol in chloroform (2 mg/lOO ml). The resulting fluorescence was measured on a spectrofluorimeter (Aminco-Bowman, Silver Spring, Maryland, U.S.A.) with excitation at 535 nm and emission 560 nm. The results were calculated by reference to aqueous estriol standards in the range 6-30 mg/2 1. (b) Gas-chromatographic manual method. The total phenolic steroids were extracted by the method of Brown [ll] after hydrolysis of urine in hydrochloric acid (15 : 100, v/v) in a boiling water bath for 30 min. The resulting extracts were evaporated to dryness, acetylated, and chromatographed at 265°C on a 5 ft glass column packed with 3.8 U.C.C.W.-982 on chromosorb G80-100 (Hewlett-Packard, Ltd, Slough, England). The internal standard was 5a-cholestan. Glucose estimations Glucose concentrations were determined by the glucose of Morley et al. [12] with guaiacum as oxygen acceptor.
Measurement
oxidase
method
of radioactivity
The P-radiation of [6,7-3H]estriol (specific measured with a Wallac 8100 liquid scintillation 5-161 25 Bromma 1, Sweden).
activity counter
of 2.5 Ci/mmol) (LKB Produkter
was AB,
199
Results
Experiments
with estriol
To determine the effect of glucose upon the estriol recovery a series of estriol solutions at a concentration of 15 mg/2 1 containing increasing concentrations of glucose between 0.02 and 0.1 M, were estimated for estriol with the automated method. A progressive decrease in recovery was found with increasing glucose concentration (Fig. 1). The effect upon estriol recovery of treating 5-ml aliquots of an estriol solution in 0.1 M glucose with increasing quantities of borohydride for one hour at 37°C is shown in Fig. 2. Excess borohydride was removed by the addition of 0.5 ml glacial acetic acid. Estriol was determined with the automated method. The concentration of glucose remaining was determined in each sample. Results show that the estriol recovery increased as the glucose concentration decreased. From these data the addition of 50 mg sodium borohydride to 5 ml of sample was judged sufficient to remove the glucose likely to be present in urine samples. Since the action of borohydride upon glucose is to reduce the aldehyde group, converting glucose to sorbitol [13], it was believed that it was the aldehyde group of the glucose molecule which was responsible for decreases in estriol recovery. To test this hypothesis the effect of a number of sugars and other substances, some having an aldehyde group, was tested. Each compound was added to a standard 15 mg/2 1 es-trio1 solution so that the final concentration of the additive was 0.1 M and estriol was estimated by the automated method. In each case the effect upon estriol recovery of pretreating 5 ml of sample with 50 mg sodium borohydride was assessed (Table I). These results demonstrated that aldehydes decrease the recovery of estriol and that in many cases this effect could be removed with borohydride.
o: 0
0.02
0.04 GLUCOSE
0.00 CONC.
Od8
0.1
(moles/L)
Fig. 1. The effect of increasing concentrations of glucose upon estriol recovery. The points represent experimmtal values. the broken line the calculated curve for the reaction of one molecule estriol with two molecules glucose.
BOROHYDRIDE
Fig. 0.1
2.
Estriol
M glucose.
recovery Final
hQ/+ml)
(0 ----a)
concentration
following of glucose
borohydride (m -9)
treatment following
of
a standard
borohydride
solution
treatment
of
estriol
in
of the standard
solution.
TABLE
I
RECOVERY
OF
ADDED
ESTRIOL
IN
THE
PRESENCE
OF
POTENTIAL
SUB-
INTERFERING
STANCES Group
A:
Compounds
Compounds
which
may
in which be present
an aldehyde
group
physiologically
is either
present
or as a result
or formed
of the
in the reactien.
Group
reaction.
~-Additive estriol
Group
to
Direct
Hydrolysis
hydrolysis
standard
following
borohydride
reduction Estriol
Estriol
cont.
rec.
Estriol
Estriol
mg/2I)
e’o)
cont.
rec.
(mg121)
(%)
15
100
A
NOIlk?
15
100
GlWXXe
2.1
14
14.5
97
Galactose
1.8
12
15.2
101
Fructose
1.1
7
14.1
94
Xylose
1.1
7
15.2
101
Lactose
1.1
7
3.0
SUClXWZ
0.6
4
1.1
Acetaldehyde
2.7
18
4.5
30
Formaldehyde
2.1
14
12.3
82
Group
20 7
B
Sorbitol
14.8
99
14.9
99
Acetone
14.1
94
13.8
92
37
14.9
99
Acetoacetate
5.5
/3-Hydroxybutyrate
13.0
94
12.7
92
Pyruvak
11.4
76
14.5
97
Lactate
13.5
90
15.2
101
Citrate
15.4
103
15.3
102
Oxalate
15.4
103
15.0
100
Hippurate
15.0
100
15.0
100
B:
201
TABLE THE
II EFFECT
UPON
ESTRIOL
EXTRACTION
OF
THE
PRESENCE
OF
ALDEHYDE
DURING
ACID
HYDROLYSIS
Sample
Net
Tritiated
estriol
standard
Tritiated
estriol
standard.
Tritiated
estriol
standard
and extracted
with
cpm
Recovery
(9%)
58558 hydrolysed
and
in formaldehyde
ether
extracted (0.1-M).
with
ether
44 422
76
37
0
hydrolysed
Since the analysis of estriol by gas chromatography involved different principles, the effect of glucose and aldehyde upon the recovery of estriol in this method was tested. Solutions of estriol were estimated without any additions, and in the presence of glucose or formaldehyde. The results were similar to those found by the automated method, with glucose causing a marked decrease and formaldehyde complete loss of the estriol. In both instances this decrease was prevented by pretreatment of the sample with borohydride. Some measurements with [ 3H] estriol were carried out in order to determine whether as a result of the reaction between estriol and an aldehyde the product remained in the aqueous acid phase. This was not practical in the automated method where the highly coloured product in the aqueous phase and the strong UV absorption of the p-nitrophenol in chloroform quenched the emission of the scintillant generated by the [ 3H] estriol, and so experiments were made with the manual method. The hydrolysis step of the manual technique did not produce any strongly coloured products; an estriol solution containing a trace quantity of the tritium labelled estriol was subjected to hydrolysis conditions and the hydrolysate extracted with ether. A duplicate estriol solution containing formaldehyde at a concentration 0.1 M was treated in the same way. The amount of radioactivity in the ether extracts was measured and compared with a standard solution which had not been processed (Table II). The results show that the radioactivity which had formerly been associated with estriol remained in the aqueous phase when formaldehyde was present during hydrolysis.
Experiments
with urine
A comparison was made of the estrogen values obtained from 28 urines which contained glucose. Each sample was analysed without any pretreatment and also after borohydride reduction. The borohydride reduction was carried out as follows: To 5 ml urine were added 0.5 ml 10% sodium borohydride in 0.1 N sodium hydroxide, and one drop of octan-2-01 to prevent the mixture frothing. The tubes were incubated for one hour at 37”C, excess borohydride was removed by acidifying with 0.5 ml glacial acetic acid and the estrogen was estimated by the automated method, due allowance being made for dilution. The mean increase in the values obtained for urinary estrogen following borohydride reduction was 9.3 mg/2 1 with a range of increase of 2.0-23.5 mg/2 1. A similar comparison was made of 111 pregnancy urines which were glucose free. The increase in urinary values is shown in Fig. 3.
202
INCREASE
Fig. 3. The effect tion.
IN
ESTROGEN
URINARY
of borohydride
treatment
(MG/ZL)
upon
“glucose
negative”
pregnancy
urines estriol determina-
Discussion The nature of the action of glucose in causing a marked decrease in urinary estrogen yield when analytical procedures are used which have an acid hydrolysis step has been shown to be due to the aldehyde group of the molecule. Studies with radioactive estriol show that the product of the reaction between estriol and an aldehyde remains water soluble after acid hydrolysis. The initial study of the reaction between estriol and varying concentrations of glucose showed a direct but nonlinear relationship between the estriol recovery and glucose concentration. These observations suggested that the reaction could not be explained on a simple mole per mole basis, and a theoretical model was considered in which one molecule of estriol reacted with two molecules of glucose according to the equations: eGandeG+G+eG,
e+G=
where E is estriol and G is glucose. reactions may be expressed as
&~[EGl
and
K
=
*
[EI [Gl
LEG2 ~ I [EGI
Then E may be expressed K, K,
[EI K,
[Gl
= LeGI,
[Gl
as follows K2
[EGI
[EI [Gl’ = [&I
%otal = Eestimated
* (1 + KlG
With equilibrmm
+ KlKzG’)
[Gl = [%I
constants
K,
and K2 the
203 Eestd ----= ‘&al
1 _~__~ 1+ K,G + K&G*
= Estriol
recovery.
By taking two recovery values from the experiment described in Fig. 1, 0.73 at 0.01 M glucose and 0.21 at 0.07 M glucose, a simultaneous equation was derived and K1 and K2 were calculated to be 34.1 and 82, respectively. From these values for K, and Kz a line representing the relationship between estriol and glucose reacting in the ratio of 1 : 2 was calculated and is given in Fig. 1. It can be seen that the line closely follows the points found by experiment. The problem of preventing this artifact was resolved by treating the samples with sodium borohydride to reduce the aldehyde to an alcohol. This reaction is slow under the conditions of the experiment [13] and excess borohydride was required to complete the removal of the glucose in a short time, so as not to delay a test where the prompt reporting of results is essential. This effect of glucose was common to all aldehydes tested and also to compounds which were converted to aldehydes under the hot acid conditions of the method. All aldehydes present at the prehydrolysis stage were effectively removed by preincubation of the sample with borohydride. The time required to effect this reduction varied with the particular aldehydes tested. The monosaccharides required one hour at 37°C to complete the removal of the aldehyde, yet the same concentration of formaldehyde or acetaldehyde required four times more borohydride and two hours incubation to remove the artifact. This necessity to increase reagents and time with these compounds may be due to an initial reaction between estriol and these compounds, perhaps to form an acetal. The acetal so formed would be stable in the alkaline medium following the addition of the borohydride, and this equilibrium would be slow to revert to the free aldehyde for reduction. The effect of the disaccharides could not be removed by the borohydride. This is due to the hydrolysis of the disaccharide molecule during the hot acid stage to form the aldehyde furfural, which then reacts with the estriol and so prevents its estimation. As lactosuria may occur in late pregnancy it may be pertinent to consider this as a possible factor where estrogen values and clinical judgements differ. It is interesting to note that certain keto acids tested affected the recovery of estriol. This may explain the anomalies that have been experienced when attempting to reproduce the poor recoveries found with diabetic samples by adding only glucose to normal urine, or explaining the differences between diabetic urines containing the same concentration of glucose [2] . What the product of the reaction is between glucose and estriol has not been identified. However, the experiment with radioactive labelled estriol showed that in the presence of formaldehyde (an aldehyde chosen because it resulted in the complete loss of estriol) a water soluble product was formed; a finding which has also been reported between estriol and glucose [4]. As a consequence of this, methods which extract estrogens from an acid hydrolysate would have a decreased yield due to the hydrophylic nature of the product. Automated methods in which there is a direct reaction between the sample and the Kober reagent are either unable to react with this product or the extraction
204
of the Kober complex into an organic medium is prevented by its water soluble nature. The result of borohydride pretreatment of urines containing glucose was a marked increase in urinary estrogen values as the preliminary experiments with estriol suggested. The comparison of borohydride-treatment and untreated samples of urines which were all negative when tested for glucose showed increases in estrogen recovery similar to the increases demonstrated by Brown and Blair [l] in their work on the hydrolysis of conjugated estrogens, when they diluted the urines tenfold. They concluded that there was a factor responsible for the loss of estrogens during acid hydrolysis which was a normal constituent of human urine. This constituent was present in male as well as female urine, and caused a decrease in recovery both at pregnancy and non-pregnancy estrogen levels. The experiments described here suggest that it is necessary to routinely pretreat all urine samples with borohydride irrespective of whether or not they contain glucose when estrogen analyses are required. This present work enables urinary estrogen assays to be carried out on urines containing glucose, without sacrificing sensitivity of assay by dilution of the urine. It further highlights the frequent poor recoveries obtained on certain non-glucose containing urines, and proposes a simple method of eliminating this problem in the majority of cases. Acknowledgements The authors wish to thank Mr J.R.P. O’Brien, Director of the Nuffield Department of Clinical Biochemistry for allowing this work to be carried out, and the many members of this Department for their advice and encouragement during the experimental work and preparation of this manuscript. References 1
J.B.
2
R.
Brown
and
H.A.F.
Blair,
Hobkirk,
A.
Alfheim
and
3
H.L.
Yousem.
4
A.E.
Scbindler,
5
J.B.
Brown.
6
E.V.
Mackay,
7
S.L.
Cohen,
8
H. Van
9
D.G.
10
M.G.
11
J.B.
D. Solomon V. S.C.
J. Clin.
D.
and
W.L.
12
G. Morley,
A.
Dawson
M. Abdel-Akher,
J.K.
and
J. 60, V.
Hamilton
Marks, and
Clin.
Smith
(1967)
Metab.
J. Obstet.
(1967)
M.A.
and
Aust.
l9,1352
Gynec.
Chem. B. Smyth,
N.Z.
95.
595
13,186 (1968)
J. Obstet.
J. Endocrinol.
Gynaec.
7, 94
47
J. Schreurs
J. Endocrinol.
Am.
Herrmann,
29,
411
Endocrinol.
(1966)
C. Anderson,
(1971)
17,
J. Clin.
Strummer.
Endocrinol.
S&zinger,
Biochem.
13
(1959)
C. Macnaughton,
G. Gardner,
(1963) (1955)
Endocrinology
and
Macafee
M.D.R. and
Metcalf, Brown,
MacLeod,
(1969)
Campbell
and
Ratanasopa
C.A.J.
Kessel.
(1958) S. Bugge.
Clin.
and
M. Versteeg,
(1969)
Chim.
Acta
32.153
Proc.
Assoc.
Clin.
Ned.
T. Verlosk.
26.415 185 (1968) F. Smith,
(1951)
J. Am.
Biochem.
Chem.
Sot.
5. 42 73,
4691
69,
81
42.
5
Clinica Chimica Acta, 60 (1975) 197-204 0 Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6931
ESTROGEN ANALYSIS CORRECTED
M. RYAN Nuffield
IN PREGNANCY
URINE:
AN ARTIFACT
and B.C. GRAY Department
(Received
October
of Clinical
Biochemistry,
Radcliffe
Infirmary,
Oxford
(U.K.)
10, 1974)
Summary The effect of glucose and certain other compounds in reducing the yield of estrogen in urine analysis has been shown to be due to the action of the aldehyde group during acid hydrolysis. A means of removing this interference with borohydride is given, and the nature of the reaction between glucose and estriol is discussed.
Introduction A frequent problem encountered in urinary estrogen assay is the incomplete recovery of estrogen following acid hydrolysis. Several workers have demonstrated a decrease in yield when estrogen conjugates have been hydrolysed in the presence of certain compounds, notably glucose [l-5], other saccharides [3,4] and some medicaments [5,6]. Although the method of enzymatic hydrolysis of the conjugates is not susceptible to this iqterference, that of acid hydrolysis remains the more common procedure. It has been shown that estrogen recovery can be increased by dilution of the urine 5-25-fold [1,2,5], or by separation of the estrogens from urine without prior hydrolysis by precipitation [7] . Recent automated methods [8,9] in which the urinary estrogens react directly with Kober reagent have also been found to give erroneously low results in the presence of these same compounds unless a large dilution step is included in the procedure. Similar problems have been experienced in the related field of urinary 17-hydroxycorticosteroid analysis when glucose was present in the urine. One solution [lo] was the addition of borohydride in excess of that required for the reduction of the 0x0 grouping, so that all the glucose present was reduced to sorbitol. It was considered that the decreased yield in the estrogen analysis might be due to the presence, or production during hydrolysis, of aldehydes, and that these aldehydes reacted with estriol to form a complex which is not
198
measured by either the Kober reaction or gas chromatography. Reduction to an alcohol with borohydride prior to hydrolysis would nullify their action. In this report the effect upon estrogen recovery of heating acidified solutions of estriol in the presence of glucose or certain other sugars, aldehydes, ketones, carboxylic acids and alcohols has been studied. The action of borohydride in removing the effect of decreased recovery has been demonstrated with most of these compounds.
Materials and Methods All 24 h specimens of urine were collected in bottles containing a small quantity of thiomersal (B.D.H. Chemicals, Ltd., Poole, England) as preservative. The volume was recorded and specimens of less than 2 1 were diluted to this volume. This had the two-fold advantage of making most urinary estrogen concentrations fall within the range of a standard calibration curve, and of simplifying the final calculations where urinary values were expressed in mg estrogen per 24 h. Urines were routinely tested for glucose with Clinistix (Ames Co., Stoke Poges, Slough, England).
Estrogen estimations (a) Automated
method. The Kober-Ittrich procedure of Campbell and Gardner [9] was modified to give the following method. Urine sampled at 0.16 ml/min was mixed with quinol (1.5 mg/lOO ml) in sulphuric acid (65 : 100, v/v) pumped at 1.6 ml/min. This mixture was heated at 120°C for 12 min, diluted with water, cooled and the Kober complex extracted into 2% w /” p-nitrophenol in chloroform (2 mg/lOO ml). The resulting fluorescence was measured on a spectrofluorimeter (Aminco-Bowman, Silver Spring, Maryland, U.S.A.) with excitation at 535 nm and emission 560 nm. The results were calculated by reference to aqueous estriol standards in the range 6-30 mg/2 1. (b) Gas-chromatographic manual method. The total phenolic steroids were extracted by the method of Brown [ll] after hydrolysis of urine in hydrochloric acid (15 : 100, v/v) in a boiling water bath for 30 min. The resulting extracts were evaporated to dryness, acetylated, and chromatographed at 265°C on a 5 ft glass column packed with 3.8 U.C.C.W.-982 on chromosorb G80-100 (Hewlett-Packard, Ltd, Slough, England). The internal standard was 5a-cholestan. Glucose estimations Glucose concentrations were determined by the glucose of Morley et al. [12] with guaiacum as oxygen acceptor.
Measurement
oxidase
method
of radioactivity
The P-radiation of [6,7-3H]estriol (specific measured with a Wallac 8100 liquid scintillation 5-161 25 Bromma 1, Sweden).
activity counter
of 2.5 Ci/mmol) (LKB Produkter
was AB,
199
Results
Experiments
with estriol
To determine the effect of glucose upon the estriol recovery a series of estriol solutions at a concentration of 15 mg/2 1 containing increasing concentrations of glucose between 0.02 and 0.1 M, were estimated for estriol with the automated method. A progressive decrease in recovery was found with increasing glucose concentration (Fig. 1). The effect upon estriol recovery of treating 5-ml aliquots of an estriol solution in 0.1 M glucose with increasing quantities of borohydride for one hour at 37°C is shown in Fig. 2. Excess borohydride was removed by the addition of 0.5 ml glacial acetic acid. Estriol was determined with the automated method. The concentration of glucose remaining was determined in each sample. Results show that the estriol recovery increased as the glucose concentration decreased. From these data the addition of 50 mg sodium borohydride to 5 ml of sample was judged sufficient to remove the glucose likely to be present in urine samples. Since the action of borohydride upon glucose is to reduce the aldehyde group, converting glucose to sorbitol [13], it was believed that it was the aldehyde group of the glucose molecule which was responsible for decreases in estriol recovery. To test this hypothesis the effect of a number of sugars and other substances, some having an aldehyde group, was tested. Each compound was added to a standard 15 mg/2 1 es-trio1 solution so that the final concentration of the additive was 0.1 M and estriol was estimated by the automated method. In each case the effect upon estriol recovery of pretreating 5 ml of sample with 50 mg sodium borohydride was assessed (Table I). These results demonstrated that aldehydes decrease the recovery of estriol and that in many cases this effect could be removed with borohydride.
o: 0
0.02
0.04 GLUCOSE
0.00 CONC.
Od8
0.1
(moles/L)
Fig. 1. The effect of increasing concentrations of glucose upon estriol recovery. The points represent experimmtal values. the broken line the calculated curve for the reaction of one molecule estriol with two molecules glucose.
BOROHYDRIDE
Fig. 0.1
2.
Estriol
M glucose.
recovery Final
hQ/+ml)
(0 ----a)
concentration
following of glucose
borohydride (m -9)
treatment following
of
a standard
borohydride
solution
treatment
of
estriol
in
of the standard
solution.
TABLE
I
RECOVERY
OF
ADDED
ESTRIOL
IN
THE
PRESENCE
OF
POTENTIAL
SUB-
INTERFERING
STANCES Group
A:
Compounds
Compounds
which
may
in which be present
an aldehyde
group
physiologically
is either
present
or as a result
or formed
of the
in the reactien.
Group
reaction.
~-Additive estriol
Group
to
Direct
Hydrolysis
hydrolysis
standard
following
borohydride
reduction Estriol
Estriol
cont.
rec.
Estriol
Estriol
mg/2I)
e’o)
cont.
rec.
(mg121)
(%)
15
100
A
NOIlk?
15
100
GlWXXe
2.1
14
14.5
97
Galactose
1.8
12
15.2
101
Fructose
1.1
7
14.1
94
Xylose
1.1
7
15.2
101
Lactose
1.1
7
3.0
SUClXWZ
0.6
4
1.1
Acetaldehyde
2.7
18
4.5
30
Formaldehyde
2.1
14
12.3
82
Group
20 7
B
Sorbitol
14.8
99
14.9
99
Acetone
14.1
94
13.8
92
37
14.9
99
Acetoacetate
5.5
/3-Hydroxybutyrate
13.0
94
12.7
92
Pyruvak
11.4
76
14.5
97
Lactate
13.5
90
15.2
101
Citrate
15.4
103
15.3
102
Oxalate
15.4
103
15.0
100
Hippurate
15.0
100
15.0
100
B:
201
TABLE THE
II EFFECT
UPON
ESTRIOL
EXTRACTION
OF
THE
PRESENCE
OF
ALDEHYDE
DURING
ACID
HYDROLYSIS
Sample
Net
Tritiated
estriol
standard
Tritiated
estriol
standard.
Tritiated
estriol
standard
and extracted
with
cpm
Recovery
(9%)
58558 hydrolysed
and
in formaldehyde
ether
extracted (0.1-M).
with
ether
44 422
76
37
0
hydrolysed
Since the analysis of estriol by gas chromatography involved different principles, the effect of glucose and aldehyde upon the recovery of estriol in this method was tested. Solutions of estriol were estimated without any additions, and in the presence of glucose or formaldehyde. The results were similar to those found by the automated method, with glucose causing a marked decrease and formaldehyde complete loss of the estriol. In both instances this decrease was prevented by pretreatment of the sample with borohydride. Some measurements with [ 3H] estriol were carried out in order to determine whether as a result of the reaction between estriol and an aldehyde the product remained in the aqueous acid phase. This was not practical in the automated method where the highly coloured product in the aqueous phase and the strong UV absorption of the p-nitrophenol in chloroform quenched the emission of the scintillant generated by the [ 3H] estriol, and so experiments were made with the manual method. The hydrolysis step of the manual technique did not produce any strongly coloured products; an estriol solution containing a trace quantity of the tritium labelled estriol was subjected to hydrolysis conditions and the hydrolysate extracted with ether. A duplicate estriol solution containing formaldehyde at a concentration 0.1 M was treated in the same way. The amount of radioactivity in the ether extracts was measured and compared with a standard solution which had not been processed (Table II). The results show that the radioactivity which had formerly been associated with estriol remained in the aqueous phase when formaldehyde was present during hydrolysis.
Experiments
with urine
A comparison was made of the estrogen values obtained from 28 urines which contained glucose. Each sample was analysed without any pretreatment and also after borohydride reduction. The borohydride reduction was carried out as follows: To 5 ml urine were added 0.5 ml 10% sodium borohydride in 0.1 N sodium hydroxide, and one drop of octan-2-01 to prevent the mixture frothing. The tubes were incubated for one hour at 37”C, excess borohydride was removed by acidifying with 0.5 ml glacial acetic acid and the estrogen was estimated by the automated method, due allowance being made for dilution. The mean increase in the values obtained for urinary estrogen following borohydride reduction was 9.3 mg/2 1 with a range of increase of 2.0-23.5 mg/2 1. A similar comparison was made of 111 pregnancy urines which were glucose free. The increase in urinary values is shown in Fig. 3.
202
INCREASE
Fig. 3. The effect tion.
IN
ESTROGEN
URINARY
of borohydride
treatment
(MG/ZL)
upon
“glucose
negative”
pregnancy
urines estriol determina-
Discussion The nature of the action of glucose in causing a marked decrease in urinary estrogen yield when analytical procedures are used which have an acid hydrolysis step has been shown to be due to the aldehyde group of the molecule. Studies with radioactive estriol show that the product of the reaction between estriol and an aldehyde remains water soluble after acid hydrolysis. The initial study of the reaction between estriol and varying concentrations of glucose showed a direct but nonlinear relationship between the estriol recovery and glucose concentration. These observations suggested that the reaction could not be explained on a simple mole per mole basis, and a theoretical model was considered in which one molecule of estriol reacted with two molecules of glucose according to the equations: eGandeG+G+eG,
e+G=
where E is estriol and G is glucose. reactions may be expressed as
&~[EGl
and
K
=
*
[EI [Gl
LEG2 ~ I [EGI
Then E may be expressed K, K,
[EI K,
[Gl
= LeGI,
[Gl
as follows K2
[EGI
[EI [Gl’ = [&I
%otal = Eestimated
* (1 + KlG
With equilibrmm
+ KlKzG’)
[Gl = [%I
constants
K,
and K2 the
203 Eestd ----= ‘&al
1 _~__~ 1+ K,G + K&G*
= Estriol
recovery.
By taking two recovery values from the experiment described in Fig. 1, 0.73 at 0.01 M glucose and 0.21 at 0.07 M glucose, a simultaneous equation was derived and K1 and K2 were calculated to be 34.1 and 82, respectively. From these values for K, and Kz a line representing the relationship between estriol and glucose reacting in the ratio of 1 : 2 was calculated and is given in Fig. 1. It can be seen that the line closely follows the points found by experiment. The problem of preventing this artifact was resolved by treating the samples with sodium borohydride to reduce the aldehyde to an alcohol. This reaction is slow under the conditions of the experiment [13] and excess borohydride was required to complete the removal of the glucose in a short time, so as not to delay a test where the prompt reporting of results is essential. This effect of glucose was common to all aldehydes tested and also to compounds which were converted to aldehydes under the hot acid conditions of the method. All aldehydes present at the prehydrolysis stage were effectively removed by preincubation of the sample with borohydride. The time required to effect this reduction varied with the particular aldehydes tested. The monosaccharides required one hour at 37°C to complete the removal of the aldehyde, yet the same concentration of formaldehyde or acetaldehyde required four times more borohydride and two hours incubation to remove the artifact. This necessity to increase reagents and time with these compounds may be due to an initial reaction between estriol and these compounds, perhaps to form an acetal. The acetal so formed would be stable in the alkaline medium following the addition of the borohydride, and this equilibrium would be slow to revert to the free aldehyde for reduction. The effect of the disaccharides could not be removed by the borohydride. This is due to the hydrolysis of the disaccharide molecule during the hot acid stage to form the aldehyde furfural, which then reacts with the estriol and so prevents its estimation. As lactosuria may occur in late pregnancy it may be pertinent to consider this as a possible factor where estrogen values and clinical judgements differ. It is interesting to note that certain keto acids tested affected the recovery of estriol. This may explain the anomalies that have been experienced when attempting to reproduce the poor recoveries found with diabetic samples by adding only glucose to normal urine, or explaining the differences between diabetic urines containing the same concentration of glucose [2] . What the product of the reaction is between glucose and estriol has not been identified. However, the experiment with radioactive labelled estriol showed that in the presence of formaldehyde (an aldehyde chosen because it resulted in the complete loss of estriol) a water soluble product was formed; a finding which has also been reported between estriol and glucose [4]. As a consequence of this, methods which extract estrogens from an acid hydrolysate would have a decreased yield due to the hydrophylic nature of the product. Automated methods in which there is a direct reaction between the sample and the Kober reagent are either unable to react with this product or the extraction
204
of the Kober complex into an organic medium is prevented by its water soluble nature. The result of borohydride pretreatment of urines containing glucose was a marked increase in urinary estrogen values as the preliminary experiments with estriol suggested. The comparison of borohydride-treatment and untreated samples of urines which were all negative when tested for glucose showed increases in estrogen recovery similar to the increases demonstrated by Brown and Blair [l] in their work on the hydrolysis of conjugated estrogens, when they diluted the urines tenfold. They concluded that there was a factor responsible for the loss of estrogens during acid hydrolysis which was a normal constituent of human urine. This constituent was present in male as well as female urine, and caused a decrease in recovery both at pregnancy and non-pregnancy estrogen levels. The experiments described here suggest that it is necessary to routinely pretreat all urine samples with borohydride irrespective of whether or not they contain glucose when estrogen analyses are required. This present work enables urinary estrogen assays to be carried out on urines containing glucose, without sacrificing sensitivity of assay by dilution of the urine. It further highlights the frequent poor recoveries obtained on certain non-glucose containing urines, and proposes a simple method of eliminating this problem in the majority of cases. Acknowledgements The authors wish to thank Mr J.R.P. O’Brien, Director of the Nuffield Department of Clinical Biochemistry for allowing this work to be carried out, and the many members of this Department for their advice and encouragement during the experimental work and preparation of this manuscript. References 1
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