PROSTAGLANDINS

Double Isotope Derivative Dilution Method for the Determination of Prostaglandin F and E Type Metabolites in Urine

Yoshio Aizawa, Kenji Yamada and Minoru Hata Department of Pharmacology, Tokyo College of Pharmacy, Hachioji-shi, Horinouchi 1432-1, Tokyo, Japan

ABSTRACT This method was developed for the quantitative determination of the main urinary metabolite (MUM) of prostaglandin (PG) F and E type in man or experimental animal. This method was based on the extraction of PG-MUM after addition of 3HPG-MUM, the esterification with "C-acetic anhydride and the separation and purification by column chromatography. Excretion values of adult men were PGF-MUM 1.46-15.01 pg/8 hr (n= ll), PGE-MUM 0.82-15.42 pg/8 hr; and with women, PGF-MUM 0.41-7.75 pg/8 hr (n=ll), PGE-MUM 0.60-11.39 pg/8 hr. ACKNOWLEDGMENT The author is very grateful to Dr. J. Pike of the Upjohn Company for the generosity with which he has presented us with the authentic material. INTRODUCTION The main urinary metabolites of PGF and PGE type have been measured by means of gas chromatography-mass spectrometry (1,2) and radioimmunoassay (3,4). On the other hand, Bojesen and Buchhave (5) reported the isotope derivative method for the determination of PGEl and PGE2 in serum using 2-amino-35S-thiazole as the reagent. Recently, we reported the determination of PGF2a and PGE2 in human cerebrospinal fluid by double isotope derivative dilution method using "C-methanol as the reagent (6). This paper describes the method for the determination of the main urinary metabolites of PGF and E type in urine by the double isotope derivative dilution method. MATERIALS AND METHODS 1) 5ar7a-dihydroxy-ll-keto-tetranorprosta-l,16-dioicacid and 7a-hydroxy-5,11-diketo-tetranorprosta-1,16-dioicacid were gifts from Dr. J. Pike of the Upjohn Company.

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3H-PGF-MUM and 'H-PGE-MUM These compoun%s were obtained from the urine of rat injected 10 lJCiof H-PGFza (2.2 Ci/m mole, New England Nuclear Corporation) or 50 ?X!iof H-PGEz (125 Ci/m mole, NENC). The urine w$s collected for 24 hr after the injection, and "HPGF-MUM or H-PGE-MUM was extracted and purified by silica gel column chromatography. Specific radioactivity of 3H-PGF-MUM and 3H-PGE-MUM were 1 Ki/Vg, 20 ?Xi/?Jg,respectively. 3) Method The urine sample was stored at -2O'C until the analysis. 100 ml of human urine or 10 ml of rat urine was used for the analysis. Approximately 6000 dpm of 'H-PGF-MUM and 3H-PGE-MUM were added to the sample of urine and adjusted to pH 3 with 6 N HCl. The urine was extracted twice with the equal volume of ether, and washed once with twice volume of 0.01 N HCl and twice volume of water. The ether extracts were evaporated to dryness under reduced pressure. The dried sample was dissolved with a small amount of toluene-ethyl acetate (9:1), and applied to silica gel column chromatography which was prepared according to the method of Sun (7). The silica gel (Wakogel C-100, Wako Pure Chemical Industry, Japan) was activated at 12O'C for 5 hr before use. The 5 g of activated silica qel was suspended in toluene-ethyl acetate (9:1), and packed into a glass tube (lx 20 cm). The column was washed.with 10 ml of toluene-ethyl acetate (9:l) and 10 ml of ethyl acetate-methanol (2O:l). The PGsMUM fraction was eluted with 20 ml of ethvl acetate-methanol (1:l). The ethyl acetate-methanol (1:l) f;action was collected and evaporated to dryness and transferred into a micro test tube (6x80 mm] with aid of a small amount of the same solvent and then dried. 0.02 ml of 14C-acetic anhydride reagent I25 mg acetic anhydride(2.5 UCi) in 1 ml of pyridine) was added into the dried sample in the micro test tube and kept in a dessicator for 24 hr at room temperature. After 24 hr, 0.1 ml of ice cold water was added into the reaction tube, and the PGs metabolite -14C-acetate was extracted three times with 0.5 ml ether. The ether layer was washed once with 3 ml of 0.01 N HCl and then evaporated to dryness. The dried sample was redissolved in a small amount of benzene and applied to the 2nd silica gel column which was prepared with 1.5 g of silica gel and benzene. The column was washed with 5 ml of benzene. The 3H-PGF-MUM-14Cacetate was eluted with 10 ml of benzene-ethyl acetate (4:1), and then the 'H-PGE-MUM-14C-acetate was eluted with 10 ml of benzene-ethyl acetate (2:3). The radioactivity of both the fractions was measured by a liquid scintillation counter. The amounts of PGF-MUM and PGE-MUM were calculated from the ratio of 3H and '"C radioactivity. 2)

RESULTS AND DISCUSSION Chromatography of acetylated PGs-MUM The chromatogram of acetylated PGs-MUM is shown in Fig. 1. 14C-Acetate of 5a,7a-dihydroxy-11-keto-tetranorprosta-1,16dioic acid was eluted with benzene-ethyl acetate (4:l) mixture and that of 7a-hydroxy-5,11-diketo-tetranorprosta-1,16-dioic 1)

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I: 5a,7a-dihydroxy-ll-keto-tetranorprosta-1,16-dioic acid (1 ug)

II: 7a-hydroxy-5,11-diketo-tetranorprosta-1,16-dioic acid (1 pg)

loo3

1

5

15 10 Test tube number B:E

-B-B:Benzene

(4:1)--r -B:E

(2:3)-

E:Ethyl acetate

Fig. 1. Silica gel column chromatography

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25

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1977 VOL. 14 NO. 6

of PGs-MUM-"C-acetate

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acid was eluted with benzene-ethyl acetate (2:3) mixture. Fig. 2 shows the chromatogram of urinary PGs-MUM acetate of rat which was injected 3H-PGF2u (2 uCi/l mg). The urine sample was collected for 24 hr and extracted without addition of 3HPGs-MUM, and analysed as 14C acetate. The radioactivity of 14C and 3H in the PGF-MUM fraction was eluted concomitantly. 2) Radiochemical purity of urinary PGs-MUM In order to ascertain the purit of the 2nd column chromatographic fraction of PGs-MUM, the Y H/14C ratio was examined after the purification by thin layer chromatography. The ratio of 3H and ?4C radioactivity in the fraction of benzene-ethyl acetate (4:l) was identical with that after the purification by thin layer chromatography, as shown in Table 1. In order to decompose the lactone, to which PGF-MUM is easily converted under acidic condition, the urine was treated with alkali according to the method of Ohki (3) and then extracted at pH 7.3, but no increase of determination value of PGs-MUM was recognized. 3) Standard curve of PGF-MUM and PGE-MUM Fig. 3 shows the standard curve for PGF-MUM and PGE-MUM. The quantity of PGs-MUM in urine can be measured down to as little as 0.5 ug with good precision by this method. 4) Excretion of PGF-MUM and PGE-MUM of rat The amounts of excreted PGF-MUM and PGE-MUM in rat urine are shown in Fig. 4. The basal excretion of PGF-MUM and PGEMUM during 6 days was 1.51-2.52 ug/day and 0.81-1.79 pg/day, respectively, PGF-MUM and PGE-MUM in rat urine were increased by administration of PGFza(l mg/rat s.c.). At 5 days after administration, the excretion was recovered to normal values. Similar results were obtained by administration of PGE2. Effect of indomethacin on urinary excretion of PGs-MUM in rat are shown in Fig. 5. PGF-MUM and PGE-MUM in rat urine were significantly decreased by administration of indomethacin (10 mg/ kg p.o.1. The content of PGs-MUM was lowest 2 days after administration of indomethacin, and the value was recovered to normal 5 days after the administration. 5) Studies in man The contents of PGF-MUM and PGE-MUM in urine of men and women were shown in Table 2. The urine was collected during the night (from 11 PM to 7 AM), The mean values of PGF-MUM and PGE-MUM in urine of men were 4.2Ok1.15 pg/8 hr and 4.79kl.46 ug/8 hr, respectively. The mean values of PGF-MUM and PGE-MUM of women were 2.18t0.66 ug/8 hr and 3.76k1.10 ug/8 hr, respectively. The results obtained by this method gave lower values as compared with those by radioimmunoassay (8). The double isotope derivative dilution method has proved to be theoretically accurate and a rapid procedure for the quantitative analysis of PGF-MUM and PGE-MUM.

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Control

3H-PGF2a

1

5

10 15 Test tube number

-B-B:E B:Benzene

(4:l)eB:E

20

25

(2:3)-

E:Ethyl acetate

Fig. 2. Column chromatogram 3H-PGF2a

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1977 VOL. 14 NO. 6

of urinary PGs-MUM of rat injected

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3H/14C 3.0 t

1

2

4 lJg

Fig. 3 Standard curve of PGF-MUM and PGE-MUM Each point represents the mean of five experiments+S.E.

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PGEz 0.5 mg

0. 8.'

PGF-MUM

6.

0.

L 6.9 PGE-MUM

4.c.

t

1 Fig. 4

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Days Urinary excretion of PGF-MUM and PGE-MUM in rat Each rat received intraperitonealy 1 mg of PGFza on the sixth day and 0.5 mg of PGEz on the twelfth day. Each bar represents the mean of five rats*S.E.

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Indomethacin

-PGF-MUM ----PGE-MUM

~g/24 hr

I I

1.0.

1

Fig. 5

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5 10 Days Effect of indomethacin on urinary excretion of PGF-MUM and PGE-MUM in rat Indomethacin(l0 mg/kg, P-0.) was administered on the fifth day. Each point 'represents the mean of five rats?S.E.

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PROSTAGLANDINS

Table

1. Radiochemical fraction

Before

purity

PGF-MUM TLC After

of 2nd column

TLC

chromatographic

Before

PGE-MUM TLC After

TLC

5.60

5.33

1

3.62

2.48

2

6.01

5.25

2

3.26

3.62

3

6.69

4.98

3

3.62

4.11

4

5.31

5.77

4

3.21

3.07-

5

6.09

5.93

5

3.02

2.92

5.94iO.46

5.45fO.34

3.34+0.23

3.24tO.56

1

AVEfS.E.

Each value activity.

indicates the ratio of SH and TLC:Thin layer chromatography

and PGE-MUM

14C radio-

Table

2. Urinary excretion of PGF-MUM subjects (ug/8 hr)

Name

Age

PGF-MUM

PGE-MUM

Name

Age

PGF-MUM

PGE-MUM

N.I.

24

2.5-l

0.93

Y.K.

24

1.16

1.56

T.Y.

23

2.00

1.18

A.I.

22

1.74

1.27

Y.O.

23

1.46

1.84

S.H.

27

0.81

1.43

T.Y.

23

3.52

3.52

Y.K.

21

1.15

9.08

K.S.

24

2.73

2.39

O.K.

23

1.00

4.50

S.Y.

23

1.76

0.82

K.H.

24

1.80

1.65

Y.A.

30

4.30

11.90

T.Y.

38

0.85

2.35

T.S.

29

5.20

15.42

M.M.

32

0.41

0.60

K.Y.

32

5.38

4.10

M.I.

25

2.66

1.04

K.T.

34

15.01

7.61

H.T.

23

1.15

6.51

M.H.

24

2.35

3.00

T.K.

51

4.70

11.39

4.20k1.15

4.79i1.46

Women

Men

AVEkS.E.

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1977 VOL. 14 NO. 6

2.18k0.66

3.76t1.10

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PROSTAGLANDINS

REFERENCES 1)

M. Hamberg and B. Samuelsson. On the metabolism of prostaglandins El and E2 in man, J. Biol. Chem., 246:6713, 1971

2)

M. Hamberg. Quantitative studies on prostaglandin synthesis in man. Anal. Biochem., 55:368, 1973 S. Ohki, Y. Nishigaki, K. Imaki, M. Kurono and F. Hirata. The levels of main urinary metabolite of prostaglandin Fla and F2a in human subjects measured by radioimmunoassay. Prostaglandins, 12:182, 1976 S. Ohki, K. Imaki and F. Hirata. Radioimmunoassay of main urinary metabolite of prostaglandin Fra. Prostaglandins, 10:549, 1975 E. Bojesen and K. Buchhave. An isotope derivative method for the analysis of prostaglandins El and E2 in serum using 2-amino- 35S-thiazole as the reagent. Biochim. Biophys. Acta., 280:614, 1972 Y. Aizawa and K. Yamada. Determination of prostaglandin Fpa and E2 contents in human cerebrospinal fluid by the radioisotope dilution method. Prostaglandins, 11:43, 1976 F. F. Sun. Metabolism of prostaglandin Fza in the rat. Biochim. Biophys. Acta., 348:249, 1974 E. Granstrom and H. Kindahl. Radioimmunoassay for urinary metabolites of prostaglandin F2a. Prostaglandins, 12:759, 1976

3)

4) 5)

6) 7) 8)

Received

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a/3/77 - Approved

11/l/77

DECEMBER

1977 VOL. 14 NO. 6