Bank et al.: Diamine oxidase in amniotic fluid 743

Eur. J. Clin. Chem. Clin. Biochem.Vol. 29, 1991, pp. 743-748© 1991 Walter de Gruyter & Co.

Berlin · New York

Diamine Oxidase Activity in Amniotic Fluid for Diagnosis of RupturedMembranes

By C. M. Bank1 *), /. P. Offermans2, A. H. Gijzen3, F. Srnits2, M. P. van Dieijen-Visser* and P. J. Brombacher3

1 Department of Clinical Chemistry, Stichting Streekziekenhuis Walcheren, Vlissingen, The Netherlands2 Department of Gynaecology, Academic Hospital Maastricht, Maastricht, The Netherlands2 Department of Clinical Chemistry, De Wever-Hospital, Heerlen, The Netherlands

(Received August 24, 1990/August 15, 1991)

Summary: Diamine oxidase in vaginal effluent is used äs a parameter for ascertaining the state of fetalmembranes. A new method using tritiated putrescine äs a Substrate is described for the determination ofdiamine oxidase in amniotic fluid and vaginal effluents. A number of tests are used for the diagnosis ofpremature rupture of fetal membranes. The described procedure for diamine oxidase activity determinationcan be used in general hospitals and has advantages over other parameters such äs pH, glucose or fructoseconcentration, and the amniotic fluid crystallization test.

Introduction

Premature rupture of fetal membranes, defined äsrupture of the membranes before onset of labor, oc-curs in 3 — 14% of all births (1 — 3). A relationshiphas been reported between premature rupture of fetalmembranes and maternal septic morbidity and mor-tality (4—6). Premature rupture is also associated withincreased incidence of amnionitis, fetal immaturityand fetal and infant morbidity and mortality (7 — 11).Management of premature rupture of fetal mem-branes is still contrpversial and diagnosis is sometimesdifficult. Many procedures have been described andare still used for the diagnosis of premature rupturepf fetal membranes, such äs measurement of pH,glucose and fruetose in the vaginal effluent, and theamniotic fluid crystallization test (fern test) (11—16).Unförtunately, none of these appears to be fülly sät-isfactory in doubtful cases. It has been reported thatamniotic fluid contains a relatively large amount ofthe enzyme diamine oxidäse (EC 1.4.3.6), which isabsent in vaginal secretions, and might be used äs amarker for premature rupture of fetal membranes (3,17 — 20). Diamine oxidase catalyses the oxidation of

*) formerly: de Wever Hospital, Heerlen.

histamine and the aliphatic diamines 1,4-diaminobu-tane (putrescine) and 1,5-diaminopentane (cadaver-ine). In human serum or plasma the diamiiie oxidaseactivity is normally low,but it is markedly increasedin pregnäncy (21). The diamine oxidase activity inamniotic fluid is variable, and at term the levels arelower than in maternal plasma (22).

The physiological role of the enzyme in pregnancyremains to be determined. Diamine oxidase is locatedin maternal decidual cells. It has been suggested thatits localisation in the placenta allows the enzyme toact äs a barrier to prevent the entry of histamine andpolyamines into the maternal circulation (28).

The increase of diamine oxidase activity during preg-nancy is thought to result from secretion or moreprobably from lysis of decidual cells with entry of theenzyme into the circulation (23). The increase has alsobeen described in neoplasia, disorders of the gastroin-testinal tract (24), allergic states (25) and cystic fibro-sis (26).In diagnosing early rupture of fetal membranes, it isoften difficult to identify the nature of the fluid in theupper vagina. Contaminants may be blood, urine,cervical mucus, which may be secreted abundantly at

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 29,1991 / No. l·!

744 Bank et al: Diamine oxidase in amniotic fluid

term, fluor or semen. It has been suggested that inthe absence of vaginal bleeding, diamine oxidase ac-tivity in the vaginal fluid can be used for the diagnosisof ruptured fetal membranes (3,17-20). The diamineoxidase activity appears to be present in the vaginaldischarge up to two hours after the appearence ofamniotic fluid in the vagina (l 8). The overall accuracyof diamine oxidase activity determinations in vaginalfluid for diagnosing the rupture of fetal membranes,in the absence of vaginal bleeding, is reported to be96-l00% (l7, 18).As vaginal fluid samples are often small in volume, asensitive analysis is required, especially for equivocalcases of suspected fetal membrane rupture. The designof a suitable assay for diamine oxidase, which isreliable and can be used in emergency situations, hasproved problematic. The spectrophotometric assay issimple to perform but requires too much sample (27).An HPLC method has been described, but is consid-ered to be elaborate and time consuming (28).

Diamine oxidase can be elegantly measured by oxi-dative deamination of [l-14C]l,4-diaminobutane or[l-14C]l,5-diaminopentane. The products from thesetwo Substrates undergo non-enzymatic cyclisation toform [14C]pyrroline or [14C]piperidine, respectively,and these can be measured after extraction in theorganic phase (29 — 31).In our study we used [3H]l,4-diaminobutane äs sub-strate. We investigated the relation of diamine oxidaseactivity in maternal serüm and amniotic fluid in re-lation to gestational age. Sensitivity and specificityfor diagnosis of the state of fetal membranes wasdetermined from frozen vaginal fluid samples of pa-tients and of a reference population.

PatientsPatients from the obstetric departments of the Academic Hos-pital Maastricht (Maastricht), De Wever-Hospital (Heerlen),and the obstetric clinic St. Elisabeth Clinic (Heerlen), wereincluded in this study. The collection of material from patientssuspected for premature rupture of fetal membranes was per-formed over a period of 18 months at these centres. Suspicionof premature rupture of fetal membranes was based on cliiiicaldata. The diagnosis of ruptured membranes was established ifthe patient had a marked continuous fluid loss and no mem-branes were feit at the first vaginal examination. The vaginalfluid sample from those patients was considered to be amnioticfluid. In contrast, in patients who delivered more than fourweeks after the initial complaints and had an uneventfül preg-nancy without any vaginal fluid loss, or intact membranes werefeit at the time of the delivery, the membranes were consideredäs intact and the vaginal fluid was considered not to be amnioticfluid.Material was collected from 120 patients (8.2% of the recordednumber of 1469 deliveries). Sufflcient Information was presentfrom 100 patients for retrospective diagnosis. These patientswere included in the study, and they revealed a prematurerupture of fetal membranes prevalence of 39%.

For determination of diagnostic capacity and reference intervalsfor diamine oxidase activity, flüids of different and knownorigin were collected. These included amniotic flujd äs well äsfluids that might be found äs vaginal effluent during pregnancy:urine, cervical mucus, fluor vaginalis or blood.

Amniotic fluid was collected from 14£ patients with amenor-rhoea of 16 to 42 weeks by amniotic puncture or by deliberatelyperforming rupture of the fetal membranes. In cases with pre-mature rupture of fetal membranes, midstream urine or catheterurine was obtained. Cervical mucus and fluor vaginalis wereobtained only when the fetal membranes were intact. All sam-ples were stored at —20 °C until examination. Gestational agewas recorded.

Materials and Methods1,4-Diaminobutane dihydrochloride (putrescine dihydrochlor-ide, Lot No. 15F-0555) and diamine oxidase (EC 1.4.3.6, 0.06U/mg at pH 7.2, 37 °C, P-7876, Lot No. 56 F-8130) wereobtained from Sigma Chemicals. [3H]l,4-diaminobutane ([2,3-3H(N)] putrescine dihydrochloride), 37 GBq/1, specific activity910 GBq/mmol, was obtained from New England Nuclear (LotNo. 2299-72).All other reagents were purchased from Merck, Darmstadt,Germany. Phosphate buffer (0.2 mol/1) was prepared by addinga solution of 0.2 mol/1 KH2PO4 to a solution of 0.2 mol/1Na2HPO4 until pH 7.40 was reached.

Alkaline buffer: to a saturated solution of NaHCO3 at roomtemperature, after filtration, NaOH (2.0 mol/1) was added untilpH 12.2 was reached.

Perchloric acid solution (1.7 mol/1) was prepared by the additionof 14.0 ml 700 g/kg HC1O4 in H2O to give a final volurne of100 ml.Substrate solution [3H]l,4-diaminobutane/l,4-diaminobutane:98.8 kBq/l//4.5 mmol/1 was prepared in phosphate buffer.

Extraction/scintillation solution was prepared by dissolving5.60 g PPO in 11 toluene. , .....

Fresh Solutions were prepared every two weeks. Protein wasdetermined by the method of Iwala et al. (32).

Radioactivity was measured with a Kontron MR 300 LiquidScintillation Counter, with automatic background substractipnof instrumental background and quench control. Counting timewas 10 minutes per sample.

Procedure

All determinations were performed on coded samples in dupli-cate. After thawing, the samples were eentrifuged for 10 minat 3000 g. Determination of diaraine oxidase activity, in samplesother than serum or amniotic fluid, was determined after sauneextraction, to avoid problems of viscosity. The samples (l vol.)were twice mixed with isotonic saline (2 vol.) and eentrifuged.The aqueous supernatants were mixed and used for the diamineoxidase assay. Protein was also determined in the extraet. Thisprocedure was also applied to samples from the patient groupsuspected of premature rupture of fetal membranes.

Diamine oxidase determination e:

To 500 phosphate buffer in a disposable tube, 200 serum,amniotic fluid or saline extraet was added and mixed. For thesample blank, 200 perchloric acid was used. A pre-mcubationwas performed in closed tube for 15 min at 37 °C The assaywas started by the addition of 50 li[(?H]l,4-diaminobutane/l,4-diaminobutane solution, the tube closed and immediately

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 29,1991 / No. 11

Bank et al: Diamine oxidase in amniotic fluid 745

mixed. After exactly 60 min at 37 °C the reaction was stoppedby the addition of 200 μΐ perchloric acid and irarnediately mixed.Serum, amniotic fluid or saline extract (200 μΐ) was added tothe sample blank.

The extraction was performed by the addition of l ml alkalinebuffer and 6 ml PPO solution. After vigorously mixing for lminute, the tubes were centrifuged for 20 min at 3000 g. Theaqueous phase was separated by freezing in an ethanol-solidCO2 mixture at — 78 °C. The organic phase was decanted intocounting vials, and the radioactivity was measured.

Within each run a blank, purified diamine oxidase solution(0.5, 1.0, 1.5, 2.0 U/l) and three quality assessment sampleswere determined. The quality assessment samples were aliquotsof amniotic fluid stored at — 20 °C.

When the coefficient of Variation for duplicate determinationswas greater than 10%, the analysis was repeated. Enzymaticactivity of the samples was calculated from Interpolation of thelinear calibration curve. Samples with diamine oxidase activityabove 2.0 U/l were diluted in saline (l : 5) and reanalysed.

0.04

0.03-

0.02

0.01-

0.00-0.075 0.000 0.075 0,150

Fig. 2. Lineweaver- urk plot for diamine oxidase in amnioticfluid. Km = 19μπκ>1/1.

Results

Optimal assay conditions for amniotic fluid

The pH Optimum for diamine oxidase determinationin amniotic fluid and for purified enzyme were deter-mined. The optimal pH for amniotic fluid was pH= 7.4. A sample size of 200 μΐ was chosen to obtainadequate accuracy and precision.

A linear relation between incubation time and meas-ured product was found (fig. 1) at a Substrate con-centration 300 μηιοΐ/ΐ. The Km, determined in a ran-dorn sample of amniotic fluid, was 19 μπιοΐ/ΐ (flg. 2).The chosen substrate concentration was 300 μιηοΐ/l(15 χ ATm), and at this substrate concentration noenzyme inhibition was fo nd. G od linearity wasfound for diamine oxidase activity in dilutions ofpurified diamine oxidase in amniotic fluid.

1100-J

80-

Χ 60-

40-

20-

10 20 30 40t [min]

50 60

Fig. 1. Diamine oxidase activity in amniotic fluid; incubationtime (minutes) versus formation of reaction prod ct(arbitrary units). Substrate concentration 300 μηιοΐ/ΐ;sample volume 200 μΐ; total volume 750 μΐ.

In the tested r nge (up to 2 U/l), good linearity fordiamine oxidase activity was also found with dilutionsof amniotic fluid and purified enzyme in saline.

Blanks and controls

Blank values were determined in each run togetherwith controls (purified enzyme preparation). Meancourit rate v lue for blanks was 2083 counts/min; dayto day Variation was 18%. "Zero activity" was definedat twice the blank count rate. This was clearly differ-ent from the count rate of the lowest control (0.27U/l), which had a mean value of 7516 counts/min;day to day Variation was 14%.

Recovery and matrix effects

Recovery of purified diamine oxidase in amniotic fluidwas 98.2% + 10 (n = 10). Recovery of amnion dia-mine oxidase in a serum pool was 99.1% ±10(n = 8).Recovery of amnion diamine oxidase in vaginal ef-fluent after saline extraction was 104.1% ±9.8(n = 11). In urine no diamine oxidase activity wasdetectable. Prior to the experiment the urines wereadjusted to pH 7.4. Recovery of diamine oxidaseactivity in urine after the addition of amniotic fluidwas always less than 100%. Figure 3 demonstratesthe inhibitory effect of urine on the enzyme activity.

Statistics

The correlation coefficient of the calibration curvesvaried from 0.950 to 0.999 (mean 0.993, n = 17).

Eun J. Clin. Chem. Clrn. Biochem. / Vol. 29,1991 / No. 11

746 Bank et al.: Diamine oxidase in amniotic fluid

1.00< l

0.20 0.40 0.60 0.80Urine in amniotic fluid, volume fraction

1.00

Fig. 3. Decrease of diamine oxidase activity in amniotic fluidupon increasing addition of urine.

Intra- and inter-run coefficients of Variation weredetermined with aliquots of the three quality assess-ment samples (tab. 1).

Tab. l. Intra- and inter-run coefficients of Variation for diamineoxidase activity determination in amniotic fluid.

Sample Intra-run (n = 20) Inter-run (n = 14)

123

Diamineoxidase(U/l)

1.181.060.27

CV(%)

3.24.39.6

Diaminef oxidase

(U/l)

1.171.040.27

CV(%)

11.013.226.4

Tab. 2. Diamine oxidase activity (mean ± SD) in amnioticfluid in relation to gestational age (total number ofpatients 145).

Amenorrhoea(weeks)

16-1920-2324-2728-3132-3536-42

Patients(n)

128

14122574

Diamine oxidaseactivity (U/l)

0.27 + 0.211.11 + 1.481.45 + 1.300.85 + 0.540.72 ± 0.760.67 ± 0.80

Reference population

Diamine oxidase activity in amniotic fluid was meas-ured in the reference population (n = 145). The re-sults in relation to gestational age are shown in table2. Highest values were found at 24—27 weeks gesta-tional age.

Diamine oxidase activity in serum was measured inan extended reference population (n = 201). The re-sults in relation to gestational age are shown intable 3.

With the exception of one fluor sample (diamineoxidase activity 1.0 U/g protein), saline extracts offluor vaginalis (n = 58) and cervical mucus (n = 15)contained no detectable diamine oxidase activity. Pro-tein was determined in amniotic fluid, vaginal fluid,cervical mucus and serum, in order to quantitate theeffluent (tab. 4).

A number of patients' urine samples (n = 167) werealso analysed; only 15 samples contained protein andall values were below 0.5 g/l. Moreover, a limitednumber (n = 15) of husband's semen samples wereanalysed, and found to contain 53.6 + 5.7 g/l protein.

Patients suspected of premature rupture offetal membranes

Diamine oxidase was determined in the vaginal fluid(with saline extraction) and expressed äs U/g protein.

Tab. 3. Diamine oxidase activity (mean ± SD) in serum inrelation to gestational age (total number of patients201).

Amenorrhoea(weeks)

16-1920-2324-2728-3132-3536-42

Patients(n)

« , .1920263285

Diamine oxidaseactivity (U/l)

0.39 ± 0.240.64 + 0.280.70 ± 0.390.76 + 0.400.67 ± 0.360.90 + 1.07

Tab. 4. Protein concentrations (mean + SD) in amniotic fluid,vaginal fluor, cervical mucus and serum during preg-nancy.

Amenor-rT"H~»<*SI

(weeks)

Protein concentration (g/l)

Amnioticfluid

16-1920-2324-2728-3132-3536-42

Number ofpatients

797542

.3

.1

.2

.3

.0

.8

±±+±-h±

129

4.96.83.72.32.11.2

Vaginal Cervicalfluor

1.11.00.70.80.83.0

±±±±±±

58

mucus*

0.9 0.61.0 -0.4 -0.6 -0.9 0.1,3.4 5.2 ± 2.4

15

Serum

636562

.7

.5

.159,85758

.6

.6

±±4-±±±

6.76.65.25.76.27.0

126

* Cervical mucus was obtained anji analysed from 15 patientsin total, of whom 13 were in the last group.

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Bank et al.: Diamine oxidase in amniotic fluid 747

Tab. 5. Sensitivity and specificity ofdiamine oxidase activity invaginal fluid for diagnosis of premature rupture of fetalmembranes.

Diamine oxidasecut-ofT value(U/g protein)

00.050.10.20.30.4

Sensitivity

(%)

74.453.841.023.17.75.1

Specificity

(%)

83.690.296.796.798.498.4

Values from 0 to l .67 U diamine oxidase per g proteinwere found. The correct diagnosis (yes/no rupturedmembranes) was detennined retrospectively. For di-agnostic purposes Receiver Operating Characteristic(ROC) curves were detennined (tab. 5, fig. 4). Theoptimal cut-off value appears to be at zero activityof diamine oxidase in the vaginal effluent: Sensitivity74.4%, specificity 83.6%. If the elevated diamine ox-idase values were due to blood in the vaginal fluid,this was confirmed by elevated protein values in theeffluent.

100-1

40 60100-Specificity [%]

80 100

Fig. 4. Receiver Operating Characteristic (ROC) of diamineoxidase for diagnosis of premature rupture of fetalmembranes (numerical data are given in table 5).

Discussion

The cliagnosis premature rupture of fetal membranesis sometimes difficult to establish. This is primarilydue to the lack of a measurable difference in thecomposition of amniotic fluid and vaginal effluent.In an attempt to overcome this problem, we deter-mined diamine oxidase in vaginal effluent. 14C-la-belled putrescine has been proposed äs a Substrate.As this label was difficult to handle in our hospitallaboratory, in contrast to routine measurements usingtritium, we introduced [3H]putrescine äs a Substrate.The intra-run reproducibility of the method was good,but the measurement technique was hampered by aninter-run coefficient of Variation, at very low enzymeactivity levels, of 26.4%. We determined the diamineoxidase activity in the vaginal effluent of 100 patientsin order to evaluate its potential äs a method in thediagnosis premature rupture of fetal membranes. Inall of these patients the state of the membranes wasconfirmed retrospectively. Prevalence of prematurerupture of fetal membranes in this population was39%. The results of this study indicate that the sen-sitivity and specificity of the diamine oxidase activitywere 74% and 84%, respectively, at a cut-off valueof zero diamine oxidase activity. Both the Sensitivityand specificity of the diamine oxidase activity deter-mination are higher than the Sensitivity and specificity(both below 70%) of pH determination and the ferntest (33), which are the most common tests for de-tecting amniotic fluid. If diamine oxidase activity wasdetected,it was then necessary to determine whetherthis was due to amniotic fluid or blood. As the proteinconcentrations in blood are much higher than in am-niotic fluid (upper limit below 10 g/l; tab. 4) the pro-tein content of the effluent plays an important rolein this differentiation.

On the basis of these results it is concluded that thedetermination of diamine oxidase activity representsa useful method for this purpose in general hospitals.However, it should be emphasized that the accuracyof 96% reported previously (17—18) could not beconfirmed.

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Prof. Dr. P. J. BrombacherDepartment of Clinical ChemistryDe Wever-HospitalP.O. Box 4446NL-6401 CX Heerlen

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