A fluorometric assay for total diamines in human urine using human placental diamine oxidase

Clinicu Chimicu Am, 112 (1981) 141- 148 * Elsevier/North-Holland Biomedical Press

CCA

141

1712

A fluorometric assay for total diamines in human urine using human placental diamine oxidase T. Matsumoto a.*, 0. Suzuki ‘, Y. Katsumata “, a M. Oya ‘, Y. Nimura a. M. Akita a and T. Hattori UFlr.~r Deparmenr Medkiw,

c$ Surgev.

md

Nagoya 466 (Jupun)

’ md

Dt~purmwnt oj l,rgul Medrbtle, h Depurtmenl

School of Medrum-.

(Received

:Vugqw

Gmrrr.~~~~’.‘?hool 01

of l.egul Mrdicrr~e. Humumu:.srr

~lunu~n~ut.su 431-31

Unrcer.s/[\~

(Jupon)

July 6th. 1980)

Summary The detailed procedure for a new fluorometric assay for total diamines in human urine is described. The diamines were purified from the urine by cation-exchange chromatography and incubated with human placental diamine oxidase. Hydrogen peroxide formed in the diamine oxidase reaction was measured fluorometrically by converting homovanillic acid to a highly fluorescent compound in the presence of peroxidase. Because of its simplicity and high sensitivity, our present method seems useful for routine clinical investigation. The data obtained from normal subjects and patients suffering from various forms of cancer are also presented.

Introduction Much interest has recently been centered around the relationship between polyamines/diamines and cancers (see reviews [l-3]). It is widely recognized that the levels of polyamines or diamines in the urine of cancer patients are generally higher than those for normal subjects. Measurements of these amines in human urine are, therefore, considered useful for the diagnosis and prognosis of cancers. Many methods have been developed for the assays of polyamines and diamines in the urine [3]. However, most of them are complicated or tedious, and thus not suitable for routine clinical investigation. In 1963 and 1966, simple enzymatic assays for polyamines using an amine oxidase were reported [4,5]. Since the elevation of diamines may be different from that of polyamines in different kinds of cancers [6], it seemed worthwhile to investigate the assay for diamines in human urine. To our knowledge, such an attempt has never been made for total diamines. In the present paper we have tried to establish a detailed procedure for a simple enzymatic assay for total

* Correspondence should Department of Surgery,

M.D.. Division of Oncology, be addressed to: Takatoshi Matsumoto, Nagoya University School of Medicine. Nagoya 466. Japan.

First

142

diamines in human urine, using placental diamine oxidase (DAO). The data obtained from normal subjects and patients suffering from various forms of cancer are also presented. Materials and methods Chemicals Homovanillic acid, putrescine-2 HCl, cadaverine-2 HCl, semicarbazide-HCl and cellulose ion exchangers (type P) were obtained from Nakarai Chemicals, Ltd., Kyoto; horseradish peroxidase (type II) from Sigma Chemical Company, St. Louis, MO; Amberlite CG-50 (type I, 100-200 mesh) from Organ0 Company, Tokyo. Other common chemicals used were of the highest purity commercially available. Urine samples From healthy subjects and in-patients suffering from various types of cancer (Nagoya University Hospital), 24-h urine samples were collected in bottles containing 20 ml of concentrated HCI. After estimation of the urine volume, part of the urine sample was stored frozen at - 20°C until analyzed. The samples were found to be stable for more than 6 months. Human placental DA0 Human placentas were obtained from the Department of Obstetrics and Gynaecology, Ekisaikai Hospital, Nagoya. They were kept frozen until used. It is advisable to check the DA0 activity of each placenta using crude homogenates and then to continue with the one showing the highest activity, since placentas vary in their DA0 activity. The amnion, the umbilical cord, the chorion and blood clots were removed from the placenta (about 300 g). It was then washed three or more times with cold saline (1 .O 1) until the material surfaces turned pink, cut into small pieces and homogenized in 300 ml of cold saline in a Waring blender for 5 min to give a crude homogenate. It was centrifuged at 18000 X g for 15 min, and the resulting supernatant was brought to 35% saturation with ammonium sulfate, followed by centrifugation (18000 X g, 15 mm). The supernatant was then brought to 65% saturation with the salt and recentrifuged (18000 X g, 15 min). The resulting pellet can be stored at 4°C for more than 3 months without any significant loss in DA0 activity. The pellet was dissolved in 9 vols of 0.01 mol/l sodium phosphate buffer (pH 6.5) and dialyzed with stirring against 50 vols (with 3 changes) of the same buffer solution for 24 h. The enzyme solution was centrifuged at 18000 X g for 15 min. The clear supernatant (10 ml) was applied to a column (0.9 cm X 3.0 cm) of the P cellulose equilibrated with 0.01 mol/l sodium phosphate buffer (pH 6.5) to remove hemoglobin. The eluted enzyme solution was used as an enzyme source. The enzyme was purified 5.4-fold to give a specific activity of 45.7 nmoljmg protein/30 min when 1 mmol/l putrescine was used as substrate. This enzyme solution was stored at 4°C until used and found to be stable for more than 1 week. When it was stored in the frozen state for a week, the activity was found to fall to 20-40%. Therefore, in order to store the enzyme for a long time without any significant loss of the activity, storage as the ammonium sulfate precipitate is preferable. In addition, after lyophilization of the enzyme, almost no significant loss in activity could be confirmed, showing that it can also be stored in the lyophilized state for a long period.

143

Preparation of Amberlite CG-SO columns Fifty grams of the Amberlite were stirred in 300 ml of distilled water for 3 min and allowed to settle for about 5 min. The supernatant was removed by aspiration. This washing procedure was repeated 10 times. The resin was then suspended in 300 ml of 3 mol/l NaOH and stirred for 2 h. The suspension was allowed to settle for 5 min and the supernatant was removed by aspiration. It was then washed 10 times, each time with 300 ml of distilled water with stirring and aspiration. The resin, which was in the sodium form, was converted to the acid form by adding 300 ml of 2 mol/l HCl, and stirred for 2 h. After settling and aspiration of the supernatant, the resin was washed with 300 ml of distilled water 10 times. Then, it was converted back to the sodium form by treatment with NaOH as described above and washed 15 times with 300 ml of distilled water. The resin was suspended in 300 ml of distilled water and the pH adjusted to 6.3 by adding glacial acetic acid. After 1 h, the pH was checked and re-adjusted to 6.3. The resin was stored at 4°C until used. Protein determination Enzyme protein was measured method. Creatinine determination Urinary creatinine concentrations Taussky [8].

by a modification

were determined

[7] of the conventional

by the method

biuret

of Bonsnes

and

Standard assay for diamines in solution The assay was carried out fluorometrically by the method of Guilbault et al. [9]. The incubation mixture contained 0.2 ml of 0.25 mol/l sodium phosphate buffer (pH 7.8) 0.1 ml of diamine solution, 0.1 ml of homovanillic acid solution (1.0 mg/ml), 0.1 ml of peroxidase solution (1.0 mg/ml) and 0.1 ml of the DA0 solution *. After incubation at 37°C for 1 h, the reaction was stopped by adding 0.1 ml of 1 mol/l NaOH solution. To the mixture was added 2.0 ml of water. The fluorescence intensity was determined with excitation at 315 nm and with emission at 425 nm using a Hitachi 650-10 S spectrofluorophotometer at 20°C. Results Standard curves The relationship between the fluorescence intensity resulting from the enzyme reaction and diamine concentrations is shown in Fig. 1. Linearity was obtained for putrescine and cadaverine over the concentration range of l.O- 15.0 nmol per assay mixture (0.6 ml). Effect of incubation time The assay mixtures containing 10 nmol of putrescine or cadaverine were incubated for various time periods. The results are shown in Fig. 2. The fluorescence intensities for both diamines reached their maximal levels at 30 min of incubation. * The activity of the eluted enzyme adjusted to be 50- 100 nmol/h/O.l

solution was measured with 1 mmol/l putrescine ml by diluting with 0.1 mol/l sodium phosphate

as substrate and buffer (pH 7.8).

144

I

I

I

2.5

5.0

7.5

~~lne-,

I

1

ID.0

(nmolei/incubation

Fig. 1. Standard curves for diamines. of the tube was 0.6 ml.

/

12.5

15.0

IIIlxtuT~)

0 ~

0, putrescine;

l -0,

cadaverine.

The final volume

Therefore, 60 mm of incubation was adopted to secure the complete consumption of diamines in the assay mixture. Since the patterns shown in Fig. 2 are dependent on the DAO activity, it is necessary to check the activity for each enzyme preparation and dilute accordingly. Substrate competition

Interference in the reaction between diamines and polyamines was checked by measuring the fluorescence resulting from the enzyme reaction in the presence of various combinations of the amines. As shown in the Table I, interference by polyamines was negligible. Nor was there mutual inhibition between putrescine and cadaverine. Interference of commonly used drugs

Various commonly used drugs were applied to the Amber&e CG-50 column (2.0

Incubation

time

imlni

Fig. 2. Effect of incubation time on the consumption of diamines. 0 cadaverine. The assay mixture (0.6 ml) contained 10 nmol of each diamine.

0, putrescine;

0 -0,

145 TABLE

I

COMPETITION The assay method

AMONG

DIAMINES

is described

AND POLYAMINES

PLACENTAL

DA0

in the text

Substrate Putrescine Cadaverine Spermidine Sperminc Putrescine Putrescine Putrescine Cadaverine Cadaverine

FOR HUMAN

Fluorescence (10 nmol) (10 nmol) (IO nmol) (IO nmol) (5 nmol) +cadaverine (5 nmol) (5 nmol) + spermidine (5 nmol) (5 nmol) + spermine (5 nmol) (5 nmol) + spermidine (5 nmol) (5 nmol) + spermine (5 nmol)

(arbitrary

unit)

100 106 2.0 I.0 101 41.1 46.6 48.9 48.9

ml), eluted with 10.0 ml of 0.5 mol/l HCl and subjected to fluorometry in the presence of 10.0 nmol putrescine as described in the standard assay. The drugs tested were: mytomycin C (amount of the drug loaded onto the column: 0.2 mg), cytarabine (2.0 mg), fluorouracil (10.0 mg), betamethasone (0.2 mg), kanamycin sulfate (20.0 mg), ampicillin (20.0 mg), oxytetracycline (20.0 mg), cefalotin sodium (20.0 mg), aminophylline (20.0 mg), ascorbic acid (20.0 mg), thiamine hydrochloride (2.0 mg), riboflavin (2.0 mg), pyridoxine (2.0 mg), phytonadione (1.0 mg), diazepam (0.2 mg), diphenhydramine (0.2 mg), chlorpheniramine maleate (0.2 mg), sulpyrine (10.0 mg), aspyrine (10.0 mg), neostigmine methylsulfate (0.05 mg), atropine sulfate (0.05 mg) and glutathione (20.0 mg). None of drugs tested gave fluorescence. Recommended procedure On the basis of the above data, we adopted the following procedure as a routine assay for total diamines in human urine. A 12.5-ml aliquot of a urine sample and 2.5 ml of concentrated HCl were placed in a test tube capped with a small glass ball and

Fig. 3. Urinary excretion of total diamines in normal subjects and cancer patients, expressed as pmol per day. ca: cancer. The solid vertical bars show the mean levels. The dotted lines represent a 2 S.D. range above and below the normal value.

146

pig. 4. Urinary exxtion of total diamlnc\ m normal SuhJcctb and ca~cc‘r pawnta. euprcwzd a nmol per mg crcatininc. ca: canccr. The solid vertical bars shou the mean Iwcls. The dotted line5 represent a Z S.D. range above and b&w the normal value. The numbcr~ of the cast\ prewntcd in Izig. 4 arc not cqual to those precntcd in Fig. 3. txcauac creatinine v.a mcasurcd not in all txw~.

heated at 100°C for 6 h in an oil bath to hydrolyze conjugated diamines; it was confirmed that all the conjugated diamines were completely hydrolyzed by this treatment. After heating, the volume of the solution was adjusted to 15 ml with distilled water. After centrifugation at 1500 X g for 10 min. 12 ml of the supernatant was adjusted to pH 5.0-7.0 with 5 mol/l NaOH and made up to 50 ml by adding distilled water, and then applied to the column (2.0 ml) of Amberlite CC-50 packed in a 3.0-ml disposable syringe. The column was washed with 15 ml of distilled water and then with 3.0 ml of 0.5 mol/l HCl. The amines were eluted with IO ml of 0.5 mol/l HCI. A O.l-ml aliquot of the eluate was placed at the bottom of a test tube and heated at 110°C for about 30 min in an oil bath to evaporate to dryness. The residue was dissolved in 0.1 ml of distilled water and subjected to the standard assay

Fig. 5. Urinac cxcrc~~o~~ of total diamlnes following surgery in stomach (0 -0) 0) patIenta. The arrow shous the time of .\urgical operation. The dotted (O7 S.D. range abo\c and hcloa the normal vclluc.

and colon cancc’r line rcpreaent a

147

as described before (the fluorescence intensity thus obtained, 1,). The blank test differed in that the mixture was incubated without the DA0 solution, which was mixed after incubation together with the 1 mol/l NaOH solution (fluorescence intensity obtained, &). As an external standard, 10.0 nmol of putrescine was added to the mixture in place of the column eluate (fluorescence intensity obtained, f, ). The blank test for the external standard was also taken by adding 0.1 ml of distilled water in place of the putrescine solution (fluorescence intensity obtained. fCh). The calculation was as follows: O.l( f, -f;,,) pmol diamines/24

X 24-h urine volume

(ml)

h= L -Lb

Recovery und reproducihilitJ Putrescine and cadaverine, 150 nmol of each, were added to urine samples (25 ml) and run through the recommended procedure. The recoveries of putrescine and cadaverine were calculated to be 99.8 and 96.6%, respectively. To check the reproducibility of the present method. urine samples of the same origin were applied to 10 different columns and subjected to the recommended procedure. As a result, the S.D. value of the variation was 5.6%. The day-to-day variation was 7.3% (mean of the measurements of 6 samples on 3 different days). Diumines in the urine of normal subjec,ts und cancer putients Using the present method, the amounts of total diamines in the urine samples of normal subjects and patients sufferin, 0 from various types of cancer were measured. The results are summarized in Figs. 3 and 4. expressed per 24 h and per mg creatinine, respectively. As shown in the figures. statistically significant differences from normal subjects were observed in various cancer groups. The percentage increase when expressed per mg creatinine was generally higher than when expressed per 24 h. Diamines in the urine following surge~\~ The amounts of total diamines were followed after surgical operation in stomach and colon cancer patients. As shown in Fig. 5, the levels of diamines in the urine tended to increase slightly, shortly after surgery, and then decreased to the normal levels in 4 weeks. Discussion In the present study, we have established a detailed procedure of the assay for total diamines in human urine. In this method, human placental DA0 was used. This enzyme has been repeatedly highly purified [lo- 131 and its properties have been well studied. In our method, we used a crude enzyme preparation only after the ammonium sulfate-precipitation in order to minimize the length of the purification procedure. This preparation was sufficient as an enzyme reagent. The human placental DA0 is highly specific for diamines such as putrescine and cadaverine, and histamine [lo- 131. In human urine, histamine is negligible comit was confirmed that pared with diamines and polyamines [ 14.151; in addition,

14x

spermidine and spermine gave negligible fluorescence (Table I). Therefore, our present assay is considered to measure a total amount of putrescine and cadaverine. As another DA0 source, the hog kidney enzyme can be mentioned [16-201 and its purified preparation is now commercially available. However, its K, values for putrescine and cadaverine are much higher than those of the placental enzyme. Thus, in our experiments, it was difficult to obtain linear standard curves with the kidney enzyme (unpublished results). The diamine levels in the urine of various cancer patients were significantly higher than those in normal subjects (Figs. 3 and 4). In addition, the levels responded well to the surgical therapy (Fig. 5). These data validate that the present assay is useful as a biochemical marker for the diagnosis of cancers and for following-up their activities. Many methods for the determination of diamines or polyamines have been developed using amino acid analyzers, thin-layer chromatography, gas chromatography, high-pressure liquid chromatography and radioimmunoassays (see review [3]). However, most of these are time-consuming or require special equipment. Antibodies specific for polyamine, which are required for radioimmunoassays, are not available for clinical use. Our present method is sensitive enough and much simpler than those so far reported. Thus, the method seems suitable for routine clinical investigation of diamines in human urine. Acknowledgements The authors encouragement

are very grateful in this study.

to Professors

Y. Iyomasa

and S. Yada for their kind

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