Changes in prostaglandin concentration in blood subjected to repetitive freezing and thawing

CHANGES IN PROSTAGLANDIN CONCENTRATION IN BLOOD SUBJECTED TO REPETITIVE FREEZING AND THAWING Morton B. Waitzman and Mary L. Law Laboratory for Ophthalmic Research, Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia 30522 ABSTRACT Successive freezing and thawing of whole blood results in a consistently higher yield of various prostaglandin (PG) compounds. Evaluations were made with radioimmunological assay. The increase in PG concentrations seems to be more associated with cell fragmentation and not with the dissociation of albumin-PG complex. Our data suggest that there may be some dissociation of non-albumin-PG complexes. Artifactually-~i~h PG concentrations due to in vitro PG synthetase activity appears minimal at least w-~th respect to indomethacin blocking of this enzyme. There are, in general, only slight differences in PG concentrations in samples with and without indomethacin.

ACKNOWLEDGEMENTS This work was supported by NIH Grants EY00243 and EY00247 and, in part, by the Southern Dames of America, Inc. We thank Dr. John Pike of the Upjohn Company, Kalamazoo, Michigan, for the gift of authentic prostaglandins.

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INTRODUCTION

After observing an increase in prostaglandin (PG) values in whole blood when it had been frozen and thawed prior to extraction compared with immediate extraction of freshly sampled blood, experiments were conducted to determine the effect of repetitive freezing and thawing on both endogenous PGs and PGs added to freshly drawn whole blood. Someevidence is provided as to the possible reasons for the effects observed.

METHODS

I.

Recovery of endogenous and exogenous PGs:

500 ml of blood from a normal human female were drawn directly into a plastic Fenwal Blood Pack into which had been injected i0 ml of a 50% alcoholic solution of heparin and indomethacin to give a final concentration of 20 USP units of heparin per ml blood and 10-4M indomethacin. The bag was kneaded as the blood was drawn into it to insure proper mixing. The fresh well-mixed blood was divided into four fractions and exogenous PGs added as shown in Table I.

TABLE I Blood Samples Analyzed for Freeze-Thaw-Recovery Blood Fraction #

950

Volume (ml)

Study

Exogenous PGs (n@/ml whole blood) FI~ F2~ E1 E2

I

120

0

O

0

0

2

60

iI

7

0.5

0

3

60

ll

7

0.5

7

4

60

0

0

0

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Duplicate 5 m! aliquots of each of the 4 fractions of blood were immediately extracted and the remaining blood stored in plastic containers in a -20°C freezer for future thawing and extraction. Aliquots of fraction #i were extracted after each of 6 successive freezings and thawings (Table II) for determination of endogenous PGs. Aliquots from fractions 2 through 4 to which exogenous PGs had been added were extracted at several designated thawings (see Table III). Because the procedures above involve the extraction of duplicate 5 ml samples of blood it was considered appropriate to confirm parallelism by extraction of a different aliquot size from the same blood pool. We therefore extracted duplicate 2.5 ml aliquots along with the 5 ml aliquots (see Table II). In one study - see Table V - multiple blood dialysate volumes ranging from 25 to 250 ~l were also assayed to confirm parallelism in this procedure. Radioimmunoassay procedures for qualitative and quantitative assessment of immunoreactive PGs were a modification of work performed in this and other laboratories (1,2). Radioimmunoassay kits (Clinical Assays, inc., Cambridge, Mass.) for detecting PGB] ~onverted from PGEI) , PGFI~ and PGF2~ were used. (It should be-pointed out that cross-reactivity of the PG antisera for these commercial kits - according to cross-reactivity curves supplied by the manufacturer - is as follows: PGFI~ anti-serum cross-reacts approximately 10% with PGF2~ , but shows only trace cross-reaction with PGBI; PGF2~ anti-serum crossreacts I-2% with PGFI~ and essentially no cross-reaction with PGBI; PGB 1 anti-serum shows no cross-reaction with either PGFI~ or PGF2~. These cross-reaction data were taken at about the 50% " T ~ a l Bound" areas of the various anti-sera standard curves. Effectively, then, the PGFI~ values of our data may be approximately 10% higher than what should be represented for PGFI~ and the values for PGF2~ are approximately 1-2% higher than what should be represented fop PGF2~). The aliquots of blood were extracted with i0 ml ETOH and allowed to stand for 1 hour before filtering. The filtrate was evaporated to dryness in a 40°C water bath under a stream of argon, and the residue taken up in Tris buffer (0.01 M, pH 7.4). The dissolved extract was then dialyzed against Tris buffer for 18 hours with a rocking apparatus (Burrell Wrist Action Shaker). The following aliquots were then taken from the 2.2 ml dialysate (pooling of duplicate l.l ml dia!ysate vessels) for immunoassay: i) 2 x 200 ~I for PGFl~ , 2) 2 x 200 ~I for PGF2~ , 3) 500 ~I were subjected to alkaline conversion of PGE 1 to PGB] by boiling in a H20 bath for 5 min after being adjusted to pH 12.5 - 12,9 with 1 N NaOH, and then determine PGBz on duplicate 200 ~i aliquots, and 4) a second 500 ~! aliquot was subjected to Na borohydride conversion of PGE 2 to PGF2~ (3). (This latter procedure for converting PGE 2 to PGF2~ has the disadvantage of resulting in a yield of 50% of the starting PGE 2. This 50% yield is a remarkably consistent yield as has been. shown by these workers (3) and as we

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have shown repeatedly in these current studies. One might resort to preliminary chromatographic separation with silver impregnated thin-layer si!icie acid plates such as that used by Goodson et al. (4) and subsequent isolation of PGE 2. We do not find this latter procedure amenable to our investigations in that; first of all, PGB 1 antibody has to be used for the evaluation of the PGE 2 and, secondly, these latter workers (6) were able to attempt this procedure because of the extremely high yield of PGE 2 in their systems compared with what one finds in blood. With reference to this latter work it should be pointed out that these workers did not do radioimmunoassay of PGE i using eiuted chromatographic substances. They were able to recover only about 39Z of the tritiated PGE 1 used as a marker on their silicic acid plates. This indicates about 2/5 loss of starting substance. Their plate recovery with respect to endogenous PGE 2 in the tissues tested is not clear, if this latter yield approaches the order of magnitude such as the 39% recovery of PGE l then it would appear more appropriate to use the Na borohydrlde conversion of PGE 2 to PGF2~ using subsequently the antibody for PGF2~. ) PGE 2 was analyzed as PGFi~ and calculated by subtracting the PGFi~ value of an aliquot not receiving borohydride treatment. In this procedure for PGE 2 analysis the necessary corrections were made for the chemical destruction of PGE 2 (as calculated from the actual loss~ during the borohydride conversion, of known amounts of PGE 2 in separate tubes). Standard curves allowed pieogram to nanogram sensitivity. (Our standard curves were based on dilutions of known amounts of PGs in a system containing known amounts of isotopic PGs. The Tris-gel buffer blank tubes which contained the isotope but no added unlabeled PG were used as the blank corrections for the standard curves. If these blank tubes were treated as though they were blood samples and carried through the total extraction procedure there was some variations in the recovery of label. The recoveries for I~F!~ , PGFi~ and PGE 1 in 20 separate studies with standard curves were 94%-+ 0 . % 9 3 ~ ~ 0.7, and 87% + 1.0 (SE), respectively. This m e a ~ t h a t ~ h e extraction procedure i~self leads to minimal false high values. The greater effect for PGE l is due likely to the eonversion of }<3El to PGB 1 through the alkalinization procedure and the accompanying destruction of some of the initial PGE 1 substance.) The antibody-bound precipitated PG which was ultimately prepared was dissolved in i ml of 0.i N acetic acid and transferred to scintillation vials and counted for i0 minutes in a Beckman-LS 250 Three Channel Ambient Liquid Scintillation Counter. II.

Effects of preincubation of blood with indomethacin on PG recovery:

Incubation of blood samples was performed in separate studies on diabetic individuals to evaluate an/effects of the

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freeze-thaw procedure on PG concentration. (Diabetic individuals were used because of available data from previous studies (i) which showed a generally higher concentration of PGs in the diabetic individuals compared with non-diabetics. This previous study did not utilize the repetitive freeze-thaw procedure described in this report.) Fresh blood samples from these latter individuals were added to tubes containing 10 -4 M indomethacin and 20 HSP units of heparin/ml blood and were divided subsequently into two parts. One part was placed immediately into a -20°C freezer and the other part incubated in a 57°C water bath for i hour and then i % too, was placed in a -20°C freezer. Subsequent handling of the two aliquots was identical - and followed the procedure of successive freezethaw manipulations as described above. III. Binding effects of albumin on PGA!: An additional experiment was designed to demonstrate i f PG may be bound to human albumin and then released by freezing and thawing five times. Two solutions were prepared containing: Tube A - Tris Buffer + cold PGA 1 + HS-PGAI Tube B - Tris Buffer + cold PGA 1 + ~ - P G A 1 + Human albumin These two solutions were allowed to stand at room temperature for 3 hours - mixing 5 or 6 times by gentle inversion for binding to occur. Aliquots from each tube were counted in the liquid scintillation counter referred to above. Other aliquots (l. 1 ml) were dialyzed against I.i ml Tris buffer (0.01 M, pH 7.4) for 18-20 hours. A portion of the dialysate and a portion of the sample side were also counted. The remaining solutions in Tubes A and B were frozen at -20oc and then thawed and refrozen. This freeze-thaw procedure was repeated 5 times and aliquots were again dialyzed and counted as previously described. The specific details of chemical and isotope additions are as follows: Tube A - 5 ml ~Pris buffer 5 ~i of a cold PGA 1 solution containing 100 ng/10 ~ (thus adding i0 ng PGA 1 per ml buffer). 25 ~i of a HS-PGAI solution (estimated approximately 50,000 dpm in 5 ~I). This amount added approximately 0.06 ng PGA 1 per ml. Tube B - The same as "A) but the Tris buffer contained 4 mg/ml human albumin(Pentex, Inc., Xankakee, Illinois) prior to adding the non-isotopic and isotopic PG.

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RESULTS With each successive thaw, an increase in the endogenous PG concentration was noted with a leveling off seeming to occur after the 3rd or 4th thaw (Fraction i of-Table i). (See Table II). The trend with PGE 2 is not asclear-perhaps because of the Na borohydride destruction of much of the PGE 2 and the resultant increase in "margin of error" in the analytical corrections.

TABLE II Endogenous Prostaglandins in Whole Blood After Repetitive Freezings and Thawings

Times Thawed

Age of Blood at time of Extraction (Wks)

ng of PG/ml Whole Blood FI~

F2~

E1

E2

Fresh

0

Trace

0

0. !

Trace

1

1

Trace

0

0.1

Trace

2

7

0.5

3.0

1.2

i.i

3

Ii

4. l

21.3

5.9

e

4

15

6.0

20.5

6.4

6.6

5

17

6.3**

22.0**

7.7**

6

20

8.6

22.0

7.9

*

15.8

E2 not determined.

** The values for F%., Fo~ and E 1 are, as is described in the "Methods" section base~ on extraction from 5 ml of whole blood. Altering the aliquot size so that 2.5 ml of blood were extracted rather than 5 m! the results for these same PGs are 7.4~ 22.0 and 8.3, respectively. Parallelism with respect to different aliquot sizes is thus demonstrated.

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Recovery of the added PGs is relatively unaffected by this freeze-thaw manipulation (Table III).

TABLE III % Recovery of Exogenous PGs in Whole Human Blood which was Subjected to Repetitive Freezings and Thawings Times Thawed

PGFI~

2

PGF2~

PGE 2

Blood Fraction (see Table I) 3 2 3 2

Fresh

85

84

93

i

83

85

85 86

2 3

PGE 1

87

84

83

91

4

51

52

88

54

72

84

55

74

55

4 5

91

The quantities of PGs added to the blood fractions were verified by direct (without going through extraction and dialysi@ radioimmunoassay of the PG mixes after properly diluting them with Tris Buffer. In this latter procedure PGE 2 was found to cross-react approximately 21% in the assay for PGE I. This value compares favorably with that referred to by Stylos et al. (5). Because of the large quantity of PGE 2 added in Fraction 3 in relation to the quantity of PGE 1 added~ recovery figures for PGE~ and PGE are based on analysis of Fractions 2 and 4~ respectively. 2 (Table IIl).

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We verified that the number of times thawed rather than the age of the blood at the time of extraction was responsible for the PG increase. Blood (60 ml) from a normal human was collected. A 50 ml portion was collected in a tube containing heparin (20 USP units/ml blood) (no indomethaein) and the other 30 ml portion was collected in a tube containing both heparin and 10 -4 M indomethaein. One aliquot from each of these portions was frozen and stored at -20oC for 5 weeks before thawing and extracting. Another aliquot from each of these two portions was subjected to weekly thawings and freezings so that when extracted at the age of 5 weeks, it had been frozen and thawed 5 times. An aliquot was removed from this latter portion after i week and analyzed. As shown in Table IV~ the PG content depended~ then, mainly upon the number of times frozen and thawed rather than upon the age of the blood. The indomethacin appeared to have minimal effect, although there did appear to be an elevation of PGE 2 in the absence of indomethacin.

TABLE IV Effect of age vs Number of Thawings on Endogenous PG in Whole Blood with and without Indomethacin

# of Times Thawed

+*

1

i

+ -

5

1

Age (Weeks)

5

5

FI~

n @ / m l whole Blood F2~ E1

0 0

0 trace

+

0

trace

0

0

-

0.3

0

0.2

0

+

4.6 5.5

18.9 19.5

5.5 6.9

-

* + = with Indomethacin;

0 0

E2 0 0

9.7 15.7

- = without Indomethacin

After observing these latter results in the presence and absence of indomethacin, it was thought that perhaps BY incubating samples of blood from diabetic patients with i0 -e M indomethacin, a greater effect might be elicited, i.e., in the nonincubated in vitro system one might observe a higher PG concentration. The average difference in PG content of the two all-

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quots did not, however, seem to reflect a consistent pattern of change in PG concentration (Table V). Again, PGE 2 seems somewhat elevated (in the non-incubated diabetic female blood) but not at a significant level. The propensity for this higherPGE 2 concentration, in the presence of indomethacin, will have to De investigated further.

TABLE V Comparison of Incubated vs Non-incubated Human Blood on PG Concentration

(ng/ml blood) ~"

PG

+*

Diabetic Female

PGFI~

-

1.9 + 0.4

3.2

+

2.1 + 0.5

3.6 + i. 5

-

14.5 + 5.0

12.5 + 1.6

+

13.2 + 3.3

15.1 + 1.8

-

5.5 + 1.0

3.7 + 0.5

+

4.4 + 0.8

5.5 + 0.9

-

35.9 + 4.7

57.0 + i0.3

+

25.5 + 4.0

40.6 + 13.1

PGF2G

PGE 1

PGE 2

Diabetic Male * *

+ 1.1

+ = Incubation at 57oc for i hour prior to successive freezings and thawings in the presence of l 0 - ~ indomethacin. - = No incubation prior to successive freezings and thawings in the presence of 10-4Mindomethacin. ** To confirm the replicability of PG assays various volumes of blood dialysate were analyzed from the whole blood used in this assay. The results were as follows: 25, 50, 1 0 4 150, 200 and 250 ~i of blood dialysate for PGFI~ yielded 2.9, 2.7, 2.8, 5.0, 3.0, and 5.0 ng/ml, respective!y; and 25, 50, 100, 150, and 200 ~i for PGE l yielded 5.7, 3.8, 5.9, 5.5 and 5.4 ng/ml, respectively. ~

Values are mean Z S.E. for four separate blood samplings from 2 to 4 weeks apart. At no time is there a significant change between "-" and "+" for a given PG. (The diabetic patients were part of a controlled study to determine effects of long-term administration of indomethacin. The details of this study will be the subject of another report.)

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The results of the effect of the freeze-thaw procedure on splitting the PGA-albumin complex are shown in Table VI. The data show that PGA 1 may be bound to human albumin in Tris Buffer (as demonstrated by dialysis); and that bound PGA 1 is not unbound by 5 successive freezings and thawings of the buffer-containing albumin.

TABLE VI Effect of Successive Freeze-Thaw Procedures on Albumin-bound PGA I

% H 5- PGA ! found after dialysis Sample Side

Dialysate

H 5 PGA I in Tris

Before

50.0

50.0

Buffer Minus

Freezin 9

50.4

49.6

Albumin

After

50.0

S0.0

Freezin~

51.0

49.0

H 5 PGA 1 in Tris

Before

79.5

20.5

Buffer Plus

Freezin@

79.5

20.5

Albumin

After

80.8

19.2

Freezin[

80.9

19.1

DISCUSSION The explanation for the increase in PG content seen in whole human blood after successive freezings and thawings is not clear at this time but may be due to a combination of factors. l) Plate!et release or synthesis of PGs: Greaves (6) found an average PG content in human whole hemolyzed blood of ll ng/ml by bioassay in terms of PGE l. Blood smears taken in this Laboratory after blood had been hemo!yzed (with distilled water) or sonicated or frozen and thawed showed the freeze-thaw

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treatment to be effective in disrupting the cellular components. Indeed, as discussed below, our results would suggest that the increased PG concentration in repeatedly frozen and thawed whole blood is a reflection of PG release following cell membrane disPuption rather than dissociation from a protein complex. Glenn (7) found 35 ng/m! PGF2@ in rat platelet-rich plasma frozen and thawed four times. Th@ platelet-free plasma contained only 1 ng/ml. PGE 2 and PGF2@ are the PGs mainly evident in platelet release (8), and these ape the PGs showing the highest concentration after freeze-thaw treatment. Perhaps our studies reflect a primary effect of PG release from platelets a n ~ possibly, release from other blood cells. 2) Release of the protein-bound PG: The possible release of the protein-bound PG by the freeze-thaw treatment should also be considered as at least a contributing factor in the PG increase. According to Unger (9) more than 99% of any PG circulating in blood would be expected to be associated with albumin up to levels of 500 ~g/ml. The relative strength of binding becomes progressively weaker as-OH groups are added to the 5 member ring (i0). The relative strength of binding of PGs may be express thus:

PGA

~

(no-OH)

PGE

~

(1-OH)

PGF

(Ref. 1%11).

(2-OH)

The poorest recovery of added PG is of the more tightly bound PGE 1 (which could reflect, in part, a tight complexing of PGE 1 and PGA 1 to albumin). Only a slight increase in its recovery is noted after freezing and thawing. Our own studies confirm the high binding affinity of albumin for PGAI; and that this complex is not readily dissociated by the multiple freeze-thaw procedure. With respect to various types of PG complexes which may be found in blood, it is clear that there are some interesting and unresolved problems. For example, the mode of extraction has been considered in various ways (including the method described in this report). Would the method of choice be to extract plasma? One group of workers states"Since PGs do not interact with blood cells, the extraction and estimation can be made using plasma, thus obviating the handling of large quantities of cellular material" (12). On the other hand another group of workers (6) states "Comparison of this" (i.e., prostaglandin-like) "activity in the cellular and plasma fractions indicates that most of the activity which we reeovered from whole blood was contained in the cellular fraction". The thesis of our report would incline toward greater agreement with these latter workers.

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5) Concerning the minimal indomethacin effect, Saksena (15) found no effect when he added the inhibitor, Naproxen, to plasma. Gler~ (7) on the other hand did achieve some inhibitory effect on platelet-rich plasma when he incubated it with indomethacin at 37°C for i hour. In our study we did not see consistent effects with similar incubation procedures in whole blood from diabetic persons except for some rise in PGE 2. The differences may relate simply to dilution effect of the whole blood with respect to platelet-rich plasma or the relatively potent plate!et PG synthetase activity. Raz et al. (14) found a partial recovery of indomethacin-inhibited PG synthetase activity after extensive dialysis. Our data (Table IV) after 5 freeze-thaw procedures would tend, at least in part (especially with respect to PGE2) , to confirm this latter. Possibly, then, (for reasons not too clear) the dialysis to which our samples are exposed tends to partially negate the indomethacin effect. Ot~explanations for minimally demonstrable indomethacin in our studies may be as follows:

effects of

a) The radioimmunoassay is measuring (cross-reacting with) something other than PG such as a PG metabolite. At least two reports mitigate against this argument at least with respect to 15-keto PGF2~ in which cross-reaction with PGF2~ anti-serum was less than 8% (15~ 16). b) Indomethacin may actually be blocking PG synthetase even under in vitro conditions, but this blocking action may be masked by some other effect. This "masking action" may b % as suggested in a recent report~ an indomethacin blocking of 15-hydroxy-PG-dehydrogenase (17). Under these conditions~ then~ is it possible to reach a metabolic "stalemate" where synthesis and destruction are blocked concurrently? This latter investigation (17) was conducted with lung tissue dehydrogenase preparations so conclusions concerning this possible effect of indomethacin on this dehydrogenase would have to be confirmed in future studies. CONCLUSIONS Because of the higher yield of PGs following repeated freeze-thaw procedures for whole blood it is recommended that this technique be utilized for PG radioimmunoassay. In studies involving extremely high yields of PGs in biological systems, i.e., such as in the assay of homogenates of gingival tissue (about 200-400 ng PGEp/g wet weight tissue (4 9 and PGs from a pathological fluid [PGE 1 equivalent as high as 800 ng/ml (18)) then it would be appropriate to confirm the amounts of PG present using procedures such as bioassay; but even with bioassay we can attempt to block only known stimulators of muscle contraetion (stimulators other than PG) - thereby unknown stimulators can give false high contraction effects.

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REFERENCES i.

Waitzman, M. B. Prostaglandins and Diabetic Retinopathy. Exp. Eye Res. 16:507-515, 1973.

2.

Levine, L., Gutierrez-Cernosek and Van Vunakis, H. Specificities of Prostaglandins BI, FI~ and F2~. AntigenAntibody Reactions. J. Biol. Chem. 246:6782-6~85, 1971.

3.

Levine, L., Hinkle~ P. M., Voelkel, E. F. and Tasjian, A.H. Prostaglandin Production by Mouse Fibrosarcoma • Cells in Culture: Inhibition by indomethacin and Aspirin. Biochem. Biophys. Res. Comm. 47:888-896, 1972.

4.

Goodson, j. M., Dewhirs% F. E. and Brunetti~ A. glandin E2 Levels and Human Periodontal Disease. Prostaqlandins 6:81-85, 1974.

5.

Stylos, W., Howard, L., Ritzi, E. and Skarnes, R. The Preparation and Characterization of Prostaglandin E 1 Anti-serum. Prostaglandins 6:1-15~ 1974.

6.

Greaves, M. W. and McDona!d-Gibson~ W. Extraction of Prostaglandin-like Activity from Whole Human Blood. Life Sci. 2(part i):75-81~ 1972.

7.

Glenn, E. M.~ Miles~ E., Wilkes~ J. and Bowman~ B. J. Plate!et% Prostaglandins, Red Cells~ Sedimentation Rates, Serum and Tissue Proteins and Non-Steroidal Antiinflammatory Drugs. Proc. Soc. Exp. Biol. Med. 141: 879-886, 1972.

8.

Silver, M. J.; Smith, J. B., Ingerman, C. and Kocsis, J.J. Human Blood Prostaglandins: Formation During Clotting. Prostaglandins ~:429-436, 1972.

9.

Unger, W. G. Binding of Prostaglandin to Human Serum Albumin. J. Pharm. Pharmacol. 2_~4:470-477~ 1972.

I0.

Attallah, A. A. and Schuss!er~ G. C. Prostaglandin Binding in Human Serum Follows the Polarity Rule. Pr0sta@landins 4:479-484~ 1973.

!l.

Raz, A. Interaction of Prostaglandins with Blood Plasma Proteins. Comparative Binding of Prostaglandins A2~ F2~ and E 2 to Human Plasma Proteins. Biochem J. 130: 631-636, 1972.

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12.

Unger, W. G.; Stamford, I. F. and Bennett, A. Extraction of Prostaglandins from Human Blood. Nature 233:556-3Z7, 1971.

13.

Saksena~ S. K. and Castracane, V. D. Validity of Prostaglandins E and F in Rabbit Blood. Prostaglandins ~:41-44~ 1975.

14.

Raz, A.~ Stern~ H. and Kenig-Wakshai; R. indomethacin and Aspirin Inhibition of Prostaglandin E 2 Synthesis by Sheep Seminal Vesicles Microsome Powder and Seminal Vesicles Slices. Pros ta[landins~:Z37-352~ 1973.

15.

Hillier, K. and Dilley~ S. R. Separation and Radioimmunoassay of F~ Prostaglandins using Silica Gel Micro Columns. Prosta@landins ~:137-150, 1974.

16.

B i t % L. Z. and Baroody, R. Concentrative Accumulation of ZH-Prostaglandins by some Rabbit Tissues in Vitro: The Chemical Nature of the Accumulated 3H-Labelled Substances. Prostag!andins ~:131-!40, 1974.

17.

Hansen, H. S. Inhibition by Indomethacin and AspiPin of !5-Hydroxy-Prostaglandin Dehydrogenase in Vitro. Prosta~landins 8:95-i05, 197¢.

18.

Jackson, R. T., Waitzman, M. B., Pickford, L. and Nathanson, S.E. Prostaglandins in Human Middle Ear Effusions. Prosta~landins i_~0:365-371, 1975.

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