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Extraction of estrogens by the hind limb of the dog. Evidence for entry into the lymphatics
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EXTRACTION OF ESTROGENS BY THE HIND LIMB OF THE DOG. ENTRY INTO THE LYMPHATICS. D.C. Collins, E.L. Bradley III,
P.I.
EVIDENCE FOR
'
Musey and J.R.K. Preedy
Departments of Surgery and Medicine, Emory University School of Medicine, Atlanta, Georgia 30322. Received: Rec'd. 5-T-77
ABSTRACT
Substantial extrasplanchnic metabolism of estrogens is known to occur in humans and dogs. As part of an investigation into the anatomic sites of such metabolism, the extraction of estrogens by the hind limb of the dog was studied during a constant infusion of [3H]estrone. Simultaneous femoral artery (A) and femoral vein (FV) plasma samples were obtained and analyzed f r total radioactivity, unconjugated and conjugated radioactivity, for [s Hlestrone and for its metabolites estradiol-178, estrone sulfate and estrone glucosiduronate. The percent extraction across the hind limb was calculated [lOO(l-FV/A)]. The mean percent extraction + SE of total, conjugated and unconjugated radioactivity was 31 + 3.9, 27 + 4.4 and 16 + 3.7 respectively, indicating significant net uptake of these moieties by the hind limb (Pc.01). Mean percent extractions * SE for estrone and estradiol-178 were 40 + 4.9 and 32 + 2.7, indicating significant net uptake of these specific unconjugated estrogens by the hind limb (Pc.01). The mean percent extraction of estrone glucosiduronate was 16 + 3.1 indicating siqnificant net uptake of this conjugate (Pc.01). However, the mean percent extraction of estrone sulfate was negative (-12 + 4.1) indicating net production of this conjugate by the hind limb (Pc.01). Since the net uptake of total radioactivity cannot be explained on the basis of metabolism by the hind limb, the lymphatics were investigated as an alternate efferent pathway. In similar experiments the thoracic duct was cannulated, the estrogens in lymph were analyzed and compared with those in femoral artery plasma. Each estrogen measured in lasma appeared in lymph within 10 minutes following the start of the [5 Hlestrone infusion. The lymph/femoral artery concentration ratios reached a plateau at 70-100 minutes after the start of the infusion. The plateau concentrations It is suggested that removal of estrowere 20-70% of those in plasma. gens in the lymph may account, in part at least, for the net uptake of total radioactivity across the hind limb calculated from the plasma data. INTRODUCTION Although the liver is usually regarded as the chief site of estrogen metabolism
in mammalian species, substantial extrasplanchic
metabolism also occurs. least 35% Of the total
In the human it has been inferred that at metabolism of estrone (2) and 15-25% of the
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456
TIlROIDS
total metabolism of estradiol-178
is extrasplanchic
(3).
In addition,
we have shown by direct means that in the dog substantial estrogen metabolism occurs at specific extrahepatic spleen and the lungs (4).
sites such as the intestine,
Longcope and co-workers have obtained evi-
dence for metabolism of estrogens by the human forearm (5). In the above experiments values for arterio-venous
(4,5) as well as in others (6-8), positive
difference
(A-V) or percent extraction
[100(1-V/A)], where A and V are concentrations tative efferent vein respectively,
in artery and represen-
have been equated with metabolism.
However, such positive values could also reflect retention unchanged within the area, or removal from the area by routes other than the efferent vein. During an investigation of the uptake of estrogens by the hind limb of the dog, we obtained evidence suggesting that either retention or removal by alternative routes might in fact be an important factor in accounting for the results obtained.
Since retention appeared somewhat
unlikely, and since the lymphatics appeared to be the only alternative route, the entry of estrogens into thoracic duct lymph was analyzed, and a study made of the dynamics involved. The percent extractions of various
es trogen moieties (e.g. total,
unconjugated and conjugated radioactiv ity ) and of individual estrogens across the hind limb of the dog follow ing an infusion of C3H]estrone, together with an analysis of estrogens in thoracic duct lymph, are reported in the present communication. MATERIAL AND METHODS Nonradioactive estrogens and estrogen conjugates were obMaterials. tained from various comner al sources. The chemical purity of the nonradioactive estrogens and estrogen conjugates was checked by chroma-
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TDEOID6
457
tography in appropriate systems (4,9). [2,4,6,7_3H]Estrone (SA=105 Ci/mmole) was obtained from New England Nuclear Co., Boston, Mass. and purified immediately before infusion as previously described (9). Operative Procedure. Male mongrel dogs (weight 14-30 kg) were anestheized with intravenous pentobarbital (25 mg/kg) as previously described (4). Catheters were placed in a femoral artery, a femoral vein and the left jugular vein. In some animals, the thoracic duct was catheterized, as follows: The thoracic duct was exposed through a left cervical incision at the junction between the jugular and subclavian veins. In order to assist cannulation, the duct was dilated by temporary occlusion of lymph flow. An 18 gauge Bardic IntracatheterR was then introduced into the distal thoracic duct, and a ligature was placed about the catheter to prevent leakage of lymph. The neck was closed and lymph was allowed to drain from the catheter by gravity into a collecting tube. One hour previously the animals had been given a 200 ml cream meal. Administration of [3H]Estrone. A loading dose was prepared by dissolving approximately 100 PCi [JH]estrone in 0.2 ml 95% aqueous ethanol and diluting to 20 ml with 0 9% W/Vsaline.The infusion was prepared by dissolving abocrt 100 pCi [3H]estrone in aqueous ethanol as above, and diluting with 45 ml 0.9% saline. The loading dose was given rapidly into the jugular vein followed immediately by a constant infusion at a rate of approximately 0.67 uCi/min as previously described (4). Simultaneous blood samples were drawn from the femoral artery and femoral vein at 35, 40 and 45 min following the start of the infusion. In those experiments where the thoracic duct was cannulated, simultaneous blood samples from the femoral artery and lymph samples from the thoracic duct were obtained at 10 minute intervals between 10 and 140 minutes. Blood samples were centrifuged immediately and the plasma stored at -20°C until analyzed. Lymph samples were stored similarly. Radioactivity Measurements. Radioactivity was measured using a Packard 3320 scintillation suectrometer with automatic external standardization. A commercial scintillation fluid (Scintiverse, Fisher Scientific), modified by the addition of methanol to make 13% V/V and Triton-X 100 to make 20% W/V to improve the aqueous solubility, was used for counting all samples. The total radioactivity in plasma was determined by counting 0.2 ml in 10 ml scintillation fluid. The radioactivity present as unconjugated estrogens was determined by extracting 0.5 ml plasma 3 times with 2 ml ether. The ether extracts were combined, evaporated to dryness in vacua and redissolved in methanol. Aliquots were added to 10 ml sc%tmion fluid and counted. The radioactivity present as conjugated estrogens was determined by measuring the radioactivity in the aqueous phase following the ether extraction. The dpm for each sample was calculated using external standard ratios and channel ratio procedures as previously described (9). Determination of Individual Estrogens. The proteins were precipitated from 1 or 2 ml plasma or lymph by adding 4 volumes of 95% aqueous ethanol. The precipitate was separated by centrifugation and washed
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TEIROIDS
with 5 ml 80% aqueous ethanol. The supernatant was dried -___ in vacua and resuspended in 0.1 M acetate buffer, pH 6.0. Unconjugated Estrone and Estradiol-17B. The resuspended extract, described above, was extracted 3 times with equal volumes of ether. The ether extract was dried in vacua redissolved in a small amount of methanol and applied to zstantthin-layer chromatography plates (ITLCSg, Gelman Instrument Co.). Chromatography to separate estrone and estradiol-176 was carried out using cyclohexane: ethyl acetate (97:3) as the solvent system. Standard preparation of nonradioactive estrone and estradiol-178 were chromatographed simultaneously and visualized with sulfuric acid to determine the location of estrone and estradiol178 on each plate. Corresponding areas containing radioactive estrone and estradiol-176 were collected and cut into small fractions. Each fraction was suspended in scintillation fluid and analyzed for radioactivity as described above. A radioactive peak in the expected location for estrone and estradiol-178 was taken to represent [3H$strone and [3H]estradiol-176 respectively. Estrone Sulfate. Enzymic hydrolysis of estrone sulfate was carried out by incubation-&f the aqueous phase from above at 37°C overnight after addition of 2 mg/ml of Mylase P (4). The mixture was then extracted 3 times with equal volumes of ether. The ether extract was evaporated and the residue chromatographed on ITLC-SG as described above for unFractions were collected and analyzed for radioconjugated estrone. activity as described above. Estrogen Glucosiduronates. The aqueous phase from above was adjusted to pH 5.0 using O.lM acetic acid. Beef liver B-glucuronidase (Worthington Biochemical Co., Freehold, N.J.) was added (600 Fishman units/ml) and incubated overnight at 37°C. The solution was then extracted with ether and subjected to chromatography on ITLC-SG as described above for estrone and estradiol-176. Fractions were collected and analyzed for radioactivity. The radioactive materials obtained from the plasma samples and presumed to be estrone and estradiol-176 respectively, were identified by procedures previously described (4,9). It should be noted that the analytical methods do not distinguish between the various glucosiduronates of estradiol-17f3. These are collectively described as "estradiol176 glucosiduronate(s)". Methodological Losses. Experiments were carried out to determine the methodological losses by adding known amounts of radioactive estrone, estradiol-176, estrone sulfate, estrone glucosiduronate, or estradiol17B-17-glucosiduronate to plasma and lymph samples which contained no radioactivity and carrying the samples through the described procedure. Mean recoveries from plasma for 10 samples run on separate days were 87% for estrone, 82% for estradiol-176, 58% for estrone sulfate and 49% for estrone glucosiduronate and for estradiol-176-17-glucosiduronate. There were no significant differences between these mean recoveries and the corresponding recoveries from lymph. No correction for methodological losses were applied to the concentrations in the tables.
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TElROXDb
459
The percent extractions and lymph: artery concentration ratios are unaffected by the methodological losses, since the losses were similar for each radioactive moiety and individual estrogen measured. Calculations and Statistical Analyses. The concentration of radioactive moieties and individual radioactive estrogens in femoral artery (A) and femoral vein (FV) were compared by calculation of the percent extraction ClOO-(l-FV/A)J It will be noted that a positive extraction indicates net uptake by the hind limb, whereas a negative extraction indicates net production. In each case the paired t-test was used to test the differences between mean percent extractions and zero. In Table 1 and 2 the mean percent extraction was calculated from all paired samples in all dogs, as indicated in the footnotes, and not from the average values for each dog given in the tables. In all cases a value for P of c.05 was taken to indicate significance. Linear regression analysis was carried out to test the significance of variations in the observed estrogen concentrations with time. RESULTS The average concentrations radioactivity
of total, unconjugated and conjugated
in femoral artery and femoral vein plasma following the
infusion of [3H] estrone in 5 dogs are given in Table 1 together with the average percent extractions across the hind limb. the average of 3 consecutive observations
in samples taken at 35, 40 The mean percent
and 45 minutes following the start of the infusion. extractions for all dogs are also given.
Each value is
The mean percent extraction
+ SE of total radioactivity was 31 + 3.9, which was significantly different from zero (Pc.01).
The mean percent extraction of t SE of un-
conjugated estrogens was 27 rt 4.4, and of conjugated estrogens was 16 + 3.7.
Each of these two values was significantly different from zero
(Pc.01). Corresponding values for estrone, estradiol-176, and estrone glucosiduronate
are given
in Table 2.
estrone sulfate
The mean percent
extractions + SE for estrone and for estradiol-176 were 40 + 4.9 and 32 + 2.7, respectively. zero.
Each mean was significantly different from
The mean percent extraction + SE for estrone glucosiduronate was
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‘P=ROID=
Table 1. Average concentrations (10-2dpm/ml) of total, unconjugated and conjugated radioactivity in femoral artery (A) and femoral vein (FV) plasma together with the percent extraction [% extr=lOO(l-FV/A)] across the hind limb in 5 dogs following a loading dose and constant infusion of [3H] estrone. Each value is the average concentration in 3 consecutive samples taken at 35, 40 and 45 minutes following the start of the infusion.
Total Dog #
A
15
292
16
Conjugated
Unconjugated
FV
% extr
176
125
29
23
266
223
16
110
40
270
187
31
236
199
16
325
321
1
199
173
13 -
158
152
4
% extr
A
FV
% extr
143
51
131
74
44
522
376
28
91
70
17
639
358
44
184
39
633
512
19
40
381
328
14 -
FV
A
Mean ++ SE ++
31 3.9
27 4.4
16 3.7
P*
c.01
c.01
c.01
++ *
calculated from 3 samples in each of 5 dogs = 15 samples probability
that the mean percent extraction differs from zero
16 + 3.1, which was also significantly different from zero.
In contrast,
the mean extraction of estrone sulfate was negative (-12 + 4.1,) which was significantly different from zero. estradiol-176
glucosiduronate(s)
The mean percent extraction
for
is not given since there was some doubt
as to whether a steady state for this compound was reached in our experiments. The variation with time in the percent extraction values at 35, 40 and 45 minutes was analyzed by regression analysis.
There was no signi-
ficant regression in any case.
However when regression analysis was
applied to the arterial and venous concentrations,
a slow but signifi-
cant increase in both arterial and venous concentrations was observed with time in most instances.
Consequently delay factors associated
with circulation through the hind limb must be considered.
Such delay
factors could affect the calculated percent extraction values (10,12). A correction can be applied if the circulation time is known (10).
Cir-
culation time through the hind limb of the dog is reported to be about 8 sets (11).
Applying the correction as previously described (12) re-
sulted in only trivial changes in the percent extraction values.
Con-
sequently these were ignored. Plots of the ratios of concentration
in thoracic duct lymph to con-
centration in femoral artery plasma versus time for the various radioactive moieties and individual estrogens are shown in Figures l-5.
All
radioactive moieties and individual estrogens measured in arterial plasma were also found in lymph. quite rapid.
Furthermore, entry into the lymph was
Substantial quantities were present within 10 min of the
start of the [3H] estrone infusion. In each case except estrone (Fig. 5), the data were fitted to the curve described by the equation [log (Z/(L/FA x 100)-l) = log X], where (L/FA x 100) is the ratio of concentration in plasma (FA) expressed as a percentage
in lymph (L) to concentration
(and therefore corresponding
to the ordinates in Figs. l-4), X is time in minutes and Z is a selected maximum.
The calculated curves are shown in each of the figures.
value for t (= regression coefficient/SE) all cases, indicating a good fit.
The
varied between 3.1 and 7.5 in
These values for t also indicate
that the regression coefficients differ from zero in each case at the
" *
P*
<:,Ol
4.9 40
c.07
2.7 32
3160
21 -
3150
4280
2270
1910 3590
1389
1125
29
42
4
668
695
13900
0 -
probability that the mean percent extraction differs from zero by chance
c.01
-12 4.1
24800
3490
10330
5280
A
11300
c.01
3.1 16
19 -
4
12
3060 23800
34
9
% extr
6800
4800
FV
Estrone glucosiduronate
-19
-19
-23
% extr
FV
A
Estrone sulfate
calculated from 3 samples in each of 5 dogs= 15 samples
!:"y+++
1980
2510
30 -
20700 14400
40
2040
2880
29
23800 17000
1060
62
1830
39
5600
14800
17
42
360 26
% extr
FV
461
44
606
11030
16
625
A
Estradiol-17g
624
46
3510
6500
15
% extr
FV
A
Dog #
Estrone
Table 2. Average concentration (dpm/ml) of radioactive estrone, estradiol-17B, estrone sulfate and estrone glucosiduronate in femoral artery (A) and femoral vein (FV) plasma, together with the percent extraction [% extr = 100 (l-FV A)] across the hind limb in 5 dogs following a loading dose and constant infusion of [sH] estrone. Each value is the average concentration in 3 consecutive samples taken at 35, 40 and 45 minutes following the start of the infusion.
S 80
0
r
463
TIIROXDS
UNCONJUGkVED
MINUTES
Fig. 1. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for total radjoactivity and for unconjugated radioactivity during an infusion of [ H] estrone Time is in minutes following the start of the infusion. ~!t$~'curve is that of the equation [log (Z/(L/FA x 100)-l) = log i';r where Z is a selected maximum and X is time in minutes.
Pc.01 level. The ratios for total radioactivity and unconjugated radioactivity are shown in Fig. 1.
For total radioactivity the L/FA ratio rose
steadily to reach a plateau at approximately the start of the infusion.
50% at 80-100 min following
The ratio was higher for the unconjugated
estrogens, reaching a plateau of 70% at about 80 min. The L/FA ratio for conjugated estrogens reached a plateau at about 45% at 60-80 min (Fig. 2).
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TXIROIDS
60 r
CONJUGA JED
20
I 40
I 60
I
80
I 100
I 120
I 140
MINUTES
Fig. 2. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for conjugated radioactivity during an infusion of C3H] estrone. Time is in minutes following the start of the infusion. The curve is described in Fig. 1.
Corresponding data for the individual conjugates estrone sulfate, estrone glucosiduronate Figs. 3-4.
and estradiol-17B glucosiduronatesare
given in
The L/FA ratio for estrone sulfate reach a plateau at about
45% at 60-80 min (Fig. 3).
Corresponding values for estrone glucosid-
uronate were 45% and 70 min, and for estradiol-17B
glucosiduronate(s)
35% and 90 min (Fig. 4). In contrast, the curve for estrone (Fig. 5) is quite different.
The
early L/FA ratios are higher (about 35%) and decrease to reach a steady state at about 60 min at a value of about 27%. drawn freehand.
The curve in Fig. 5 is
The higher values at the earlier times may reflect the
465
60-
50-
40: x 30e -I
20-
10 -
I
I
I
I
I
I
I
I
20
40
60
80
100
120
140
MINUTES
Fig. 3. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for radioactive estrone sulfate during an infusion of C3H] estrone. Time is minutes following the start of the infusion. The curve is described in Fig. 1.
prior administration
of the loading dose together with delay factors
associated with entry into the lymph. The average flow of lymph through the thoracic duct during these experiment was 0.48 ml per minute.
Average flow of total radioactivity
through the duct was 2.4% of the rate of infusion. DISCUSSION As previously mentioned, a positive percent extraction where A and V are plasma concentrations
([100(1-V/A)],
in artery and representative
efferent vein respectively, may be due to one or more of the following: (1) metabolism within the area drained by the vein, (2) retention in the
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466
TXIROXDB
60 ESTROIVE
GLUCOS~OU~?OIVA~-E
20
I
I
I
I
I
40
60
60
100
120
I 140
MINUTES
Fig. 4. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for radioactive estrone glucosiduronate and estradiol-17f+glucosiduronte(s) during an infusion of [3H] estrone. Time is in minutes following the start of the infusion. The curve is described in Fig. 1.
area unchanged, (3) removal from the area by a route other than the representative metabolism
vein.
Consequently, extraction can be only equated with
if significant contribution by the other two processes can
be excluded.
This in turn can only be accomplished
state) total radioactivity
if (in the steady
in venous plasma is equal to total radio-
activity in arterial plasma (i.e. extraction of total radioactivity
is
zero), since if metabolism were the only process, then radioactive products should be equivalent to radioactive precursors and total radioactivity would remain unchanged.
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TDIR.OXDDII
I 40
I 60
467
50-
IO-
I 20
I 80
1 100
120
I 140
MINUTES
Fig. 5. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for radioactive estrone during an infusion of [3H] estrone. Time is minutes following the start of the infusion. The curve is drawn freehand.
In the present report substantial uptake of total radioactivity by the hind limb of the dog was observed (mean extraction 31%, Table 1). Consequently none of the positive extraction values for unconjugated or conjugated estrogens
(Table 1) or for the various individual estrogens
(Table 2) can be equated solely with metabolism. Thenet uptake (positive extraction) of estrone and estradiol-176 by the hind limb of the dog (Table 2) corresponds with the findings in the human forearm reported by Longcope et al. (5), using similar principles but more sophisticated techniques involving double-label procedures.
Such uptake was equated
with metabolism by these authors, although the extraction of total
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TIIROXDIB
radioactivity by the forearm (if any) was not reported. quantitation of the
metabolism of estrogens
Their
by the forearm
might not be justified if significant extraction of total radioactivity were present. With regard to estrone sulfate, there was evidence for small but significant net formation of this conjugate by the hind limb (mean extraction -12%, Table 2). Longcope et al. reported variable results for estrone sulfate in the human forearm (5).
Some uptake appeared to occur
in two of five experiments, and conversion of estrone to estrone sulfate occurred in one of three experiments. The uptake of estrone glucosiduronate
(mean extraction 16%, Table 2)
by the hind limb of the dog is in contrast to the net release of estrone sulfate. workers
Estrone glucosiduronate was not studied by Longcope and co(5).
For the reasons given above, the interpretation of the uptake of estrone, estradiol-176 and estrone glucosiduronate the dog noted in these experiments
is difficult.
by the hind limb of Some metabolism would
be expected on general grounds and from previously published -___ in vivo work on the metabolism of estrogens and androgens by muscle and fat (5-8) in various species.
Our present evidence for the net formation of
estrone sulfate and the evidence from the work of Longcope et al. for the interconversion of estrone and estradiol-176 (5) would support this view.
in the human forearm
On the other hand, our finding of substan-
tial uptake of total radioactivity by the hind limb must indicate either some retention unchanged in the area, or some removal by a route other than the femoral vein, or both. Retention seems unlikely.
The loading dose would be expected to
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TPROXD= The extraction of
occupy rapidly most available storage sites.
total radioactivity did not change substantially between 10 and 45 min It would seem unlikely that storage would con-
(60 min in two dogs).
tinue at the same rate over this period of time. take up estrogens preferentially
Adipose tissue may
(14, 15) but there are no extensive
deposits of fat in the dog hind limb (in contrast to the splanchnic area, for instance).
However, it is clear that retention cannot be
completely excluded. The alternative explanation, namely, removal by some route other than the vein, appears more likely. be the lymphatics.
The only such route would appear to
The data in Figs. 1-5 and Table 3 indicate that
estrogens do in fact enter the lymphatics promptly and in substantial concentration after infusion of [3H] estrone.
So far as the authors
are aware, this is the first published study of estrogens in lymph in any species. Whether the entry of estrogens into lymph can account for the extraction of total radioactivity found across the hind limb in the present study in not yet clear.
Thoracic duct lymph,studied here, drains much
of the body and the concentrations
found in the duct may be higher or
lower than those in the lymphatics draining the hind limb. the quantitative
Furthermore,
importance of the lymphatic route will depend upon
lymph flow through the hind limb as well as lymph concentration. some explanation
However
is needed for the substantial extraction of total radio-
activity by the hind limb.
Entry into the lymphatics appears to be the
best explanation at the present time.
470
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TIlROIDb
ACKNOWLEDGEMENTS Dr. D.C. Collins is the recipient of USPHS Career Research Development Award 5-K04-AM-70381 from the National Institute of Arthritis, Metabolism and Digestive Diseases. This research was supported by NIH Grant 5-ROl-AM-02221 and 5-ROl-AM-13468. The able technical assistance of Hugh Robinson, Robinette King and Roy Weems is gratefully acknowledged. REFERENCES 1. Address reprint requests to Dr. D.C. Collins, Department of Medicine, Emory University School of Medicine, 69 Butler St., S.E., Atlanta, Georgia 30303. 2. The following trivial names and abbreviations for steroids have been used. estrone (E )=3-hydroxyestra-1,3,5(10)-trien-17-one; estradiol- 1 7B(E2)=estra-l,3,5(lO)-triene-3,17@-diol; estrone glucosiduronate (ElG)=17-oxoestra-1,3,5(10)-trien-3-yl-B-Dglucopyranosiduronate; estradio1-1713-g1ucosiduronate-17B-hydroxyestra-1,3,5(10)-trien-3-y1R-D-glucopyranosiduronate and 3-hydroxyestra-1,3,5(10)-trien-17Byl-P-D-glucopyranosiduronate; estrone sulfate (ElS)=l7-oxoestra-1,3,5(10)-trien-3-yl-sulfate. J. CLIN. INVEST. -47:93, 3. Longcope, C., Layne, C.S. and Tait, J.F., (1968). 4. Collins, D.C., Robinson, H.D., Howard, C.M. and Preedy, J.R.K., J. CLIN. INVEST. 49:2326 (1970). 5. Longcope, C., Pratt, J.H., Schneider, S.H. and Fineberg, S.E., J. CLIN. ENDOCRINOL. METAB. 43:1134 (1976). 6. Chapdelaine, A.. J. CLIN.INVEST. 48:2063 (1969). A., REV. CAN. BIOL. 31:135 7. Bleau, G., Roberts, K.D. and Chapdsaine, (1972). Schneider, S.H. and Fineberg, S.E., 8. Longcope, C., Pratt, J.H., STEROIDS 28:521 (1976). 9. Collins, KC., STEROIDS 18:341 (1971). 10. Zierler, K.L., J. CLIN. NVEST. 36:2111 (1961). AND CIRCULATION. 11. Altman. P.L. and Dittmer, D.C., FSPIRATION Federation of American Societies of Experimental Biology, Bethesda, Md. 1971, p. 414. 12. Collins, D.C., Balikian, H.M. and Preedy, J.R.K., ENDOCRINOL. 96: 1543 (1975). 13. Collins, D.C., Balikian, H.M. and Preedy, J.R.K., ENDOCRINOL. 99:420 (1976). 14. Balikian, H., Southerland, J., Howard, C.M. and Preedy, J.R.K., ENDOCRINOL. 82:500 (1964). 15. Bleau, G.. Roberts, K.D. and Chapdelaine, A., J. CLIN. ENDOCRINOL. METAB. 39:236 (1976).
455
EXTRACTION OF ESTROGENS BY THE HIND LIMB OF THE DOG. ENTRY INTO THE LYMPHATICS. D.C. Collins, E.L. Bradley III,
P.I.
EVIDENCE FOR
'
Musey and J.R.K. Preedy
Departments of Surgery and Medicine, Emory University School of Medicine, Atlanta, Georgia 30322. Received: Rec'd. 5-T-77
ABSTRACT
Substantial extrasplanchnic metabolism of estrogens is known to occur in humans and dogs. As part of an investigation into the anatomic sites of such metabolism, the extraction of estrogens by the hind limb of the dog was studied during a constant infusion of [3H]estrone. Simultaneous femoral artery (A) and femoral vein (FV) plasma samples were obtained and analyzed f r total radioactivity, unconjugated and conjugated radioactivity, for [s Hlestrone and for its metabolites estradiol-178, estrone sulfate and estrone glucosiduronate. The percent extraction across the hind limb was calculated [lOO(l-FV/A)]. The mean percent extraction + SE of total, conjugated and unconjugated radioactivity was 31 + 3.9, 27 + 4.4 and 16 + 3.7 respectively, indicating significant net uptake of these moieties by the hind limb (Pc.01). Mean percent extractions * SE for estrone and estradiol-178 were 40 + 4.9 and 32 + 2.7, indicating significant net uptake of these specific unconjugated estrogens by the hind limb (Pc.01). The mean percent extraction of estrone glucosiduronate was 16 + 3.1 indicating siqnificant net uptake of this conjugate (Pc.01). However, the mean percent extraction of estrone sulfate was negative (-12 + 4.1) indicating net production of this conjugate by the hind limb (Pc.01). Since the net uptake of total radioactivity cannot be explained on the basis of metabolism by the hind limb, the lymphatics were investigated as an alternate efferent pathway. In similar experiments the thoracic duct was cannulated, the estrogens in lymph were analyzed and compared with those in femoral artery plasma. Each estrogen measured in lasma appeared in lymph within 10 minutes following the start of the [5 Hlestrone infusion. The lymph/femoral artery concentration ratios reached a plateau at 70-100 minutes after the start of the infusion. The plateau concentrations It is suggested that removal of estrowere 20-70% of those in plasma. gens in the lymph may account, in part at least, for the net uptake of total radioactivity across the hind limb calculated from the plasma data. INTRODUCTION Although the liver is usually regarded as the chief site of estrogen metabolism
in mammalian species, substantial extrasplanchic
metabolism also occurs. least 35% Of the total
In the human it has been inferred that at metabolism of estrone (2) and 15-25% of the
S
456
TIlROIDS
total metabolism of estradiol-178
is extrasplanchic
(3).
In addition,
we have shown by direct means that in the dog substantial estrogen metabolism occurs at specific extrahepatic spleen and the lungs (4).
sites such as the intestine,
Longcope and co-workers have obtained evi-
dence for metabolism of estrogens by the human forearm (5). In the above experiments values for arterio-venous
(4,5) as well as in others (6-8), positive
difference
(A-V) or percent extraction
[100(1-V/A)], where A and V are concentrations tative efferent vein respectively,
in artery and represen-
have been equated with metabolism.
However, such positive values could also reflect retention unchanged within the area, or removal from the area by routes other than the efferent vein. During an investigation of the uptake of estrogens by the hind limb of the dog, we obtained evidence suggesting that either retention or removal by alternative routes might in fact be an important factor in accounting for the results obtained.
Since retention appeared somewhat
unlikely, and since the lymphatics appeared to be the only alternative route, the entry of estrogens into thoracic duct lymph was analyzed, and a study made of the dynamics involved. The percent extractions of various
es trogen moieties (e.g. total,
unconjugated and conjugated radioactiv ity ) and of individual estrogens across the hind limb of the dog follow ing an infusion of C3H]estrone, together with an analysis of estrogens in thoracic duct lymph, are reported in the present communication. MATERIAL AND METHODS Nonradioactive estrogens and estrogen conjugates were obMaterials. tained from various comner al sources. The chemical purity of the nonradioactive estrogens and estrogen conjugates was checked by chroma-
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TDEOID6
457
tography in appropriate systems (4,9). [2,4,6,7_3H]Estrone (SA=105 Ci/mmole) was obtained from New England Nuclear Co., Boston, Mass. and purified immediately before infusion as previously described (9). Operative Procedure. Male mongrel dogs (weight 14-30 kg) were anestheized with intravenous pentobarbital (25 mg/kg) as previously described (4). Catheters were placed in a femoral artery, a femoral vein and the left jugular vein. In some animals, the thoracic duct was catheterized, as follows: The thoracic duct was exposed through a left cervical incision at the junction between the jugular and subclavian veins. In order to assist cannulation, the duct was dilated by temporary occlusion of lymph flow. An 18 gauge Bardic IntracatheterR was then introduced into the distal thoracic duct, and a ligature was placed about the catheter to prevent leakage of lymph. The neck was closed and lymph was allowed to drain from the catheter by gravity into a collecting tube. One hour previously the animals had been given a 200 ml cream meal. Administration of [3H]Estrone. A loading dose was prepared by dissolving approximately 100 PCi [JH]estrone in 0.2 ml 95% aqueous ethanol and diluting to 20 ml with 0 9% W/Vsaline.The infusion was prepared by dissolving abocrt 100 pCi [3H]estrone in aqueous ethanol as above, and diluting with 45 ml 0.9% saline. The loading dose was given rapidly into the jugular vein followed immediately by a constant infusion at a rate of approximately 0.67 uCi/min as previously described (4). Simultaneous blood samples were drawn from the femoral artery and femoral vein at 35, 40 and 45 min following the start of the infusion. In those experiments where the thoracic duct was cannulated, simultaneous blood samples from the femoral artery and lymph samples from the thoracic duct were obtained at 10 minute intervals between 10 and 140 minutes. Blood samples were centrifuged immediately and the plasma stored at -20°C until analyzed. Lymph samples were stored similarly. Radioactivity Measurements. Radioactivity was measured using a Packard 3320 scintillation suectrometer with automatic external standardization. A commercial scintillation fluid (Scintiverse, Fisher Scientific), modified by the addition of methanol to make 13% V/V and Triton-X 100 to make 20% W/V to improve the aqueous solubility, was used for counting all samples. The total radioactivity in plasma was determined by counting 0.2 ml in 10 ml scintillation fluid. The radioactivity present as unconjugated estrogens was determined by extracting 0.5 ml plasma 3 times with 2 ml ether. The ether extracts were combined, evaporated to dryness in vacua and redissolved in methanol. Aliquots were added to 10 ml sc%tmion fluid and counted. The radioactivity present as conjugated estrogens was determined by measuring the radioactivity in the aqueous phase following the ether extraction. The dpm for each sample was calculated using external standard ratios and channel ratio procedures as previously described (9). Determination of Individual Estrogens. The proteins were precipitated from 1 or 2 ml plasma or lymph by adding 4 volumes of 95% aqueous ethanol. The precipitate was separated by centrifugation and washed
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TEIROIDS
with 5 ml 80% aqueous ethanol. The supernatant was dried -___ in vacua and resuspended in 0.1 M acetate buffer, pH 6.0. Unconjugated Estrone and Estradiol-17B. The resuspended extract, described above, was extracted 3 times with equal volumes of ether. The ether extract was dried in vacua redissolved in a small amount of methanol and applied to zstantthin-layer chromatography plates (ITLCSg, Gelman Instrument Co.). Chromatography to separate estrone and estradiol-176 was carried out using cyclohexane: ethyl acetate (97:3) as the solvent system. Standard preparation of nonradioactive estrone and estradiol-178 were chromatographed simultaneously and visualized with sulfuric acid to determine the location of estrone and estradiol178 on each plate. Corresponding areas containing radioactive estrone and estradiol-176 were collected and cut into small fractions. Each fraction was suspended in scintillation fluid and analyzed for radioactivity as described above. A radioactive peak in the expected location for estrone and estradiol-178 was taken to represent [3H$strone and [3H]estradiol-176 respectively. Estrone Sulfate. Enzymic hydrolysis of estrone sulfate was carried out by incubation-&f the aqueous phase from above at 37°C overnight after addition of 2 mg/ml of Mylase P (4). The mixture was then extracted 3 times with equal volumes of ether. The ether extract was evaporated and the residue chromatographed on ITLC-SG as described above for unFractions were collected and analyzed for radioconjugated estrone. activity as described above. Estrogen Glucosiduronates. The aqueous phase from above was adjusted to pH 5.0 using O.lM acetic acid. Beef liver B-glucuronidase (Worthington Biochemical Co., Freehold, N.J.) was added (600 Fishman units/ml) and incubated overnight at 37°C. The solution was then extracted with ether and subjected to chromatography on ITLC-SG as described above for estrone and estradiol-176. Fractions were collected and analyzed for radioactivity. The radioactive materials obtained from the plasma samples and presumed to be estrone and estradiol-176 respectively, were identified by procedures previously described (4,9). It should be noted that the analytical methods do not distinguish between the various glucosiduronates of estradiol-17f3. These are collectively described as "estradiol176 glucosiduronate(s)". Methodological Losses. Experiments were carried out to determine the methodological losses by adding known amounts of radioactive estrone, estradiol-176, estrone sulfate, estrone glucosiduronate, or estradiol17B-17-glucosiduronate to plasma and lymph samples which contained no radioactivity and carrying the samples through the described procedure. Mean recoveries from plasma for 10 samples run on separate days were 87% for estrone, 82% for estradiol-176, 58% for estrone sulfate and 49% for estrone glucosiduronate and for estradiol-176-17-glucosiduronate. There were no significant differences between these mean recoveries and the corresponding recoveries from lymph. No correction for methodological losses were applied to the concentrations in the tables.
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TElROXDb
459
The percent extractions and lymph: artery concentration ratios are unaffected by the methodological losses, since the losses were similar for each radioactive moiety and individual estrogen measured. Calculations and Statistical Analyses. The concentration of radioactive moieties and individual radioactive estrogens in femoral artery (A) and femoral vein (FV) were compared by calculation of the percent extraction ClOO-(l-FV/A)J It will be noted that a positive extraction indicates net uptake by the hind limb, whereas a negative extraction indicates net production. In each case the paired t-test was used to test the differences between mean percent extractions and zero. In Table 1 and 2 the mean percent extraction was calculated from all paired samples in all dogs, as indicated in the footnotes, and not from the average values for each dog given in the tables. In all cases a value for P of c.05 was taken to indicate significance. Linear regression analysis was carried out to test the significance of variations in the observed estrogen concentrations with time. RESULTS The average concentrations radioactivity
of total, unconjugated and conjugated
in femoral artery and femoral vein plasma following the
infusion of [3H] estrone in 5 dogs are given in Table 1 together with the average percent extractions across the hind limb. the average of 3 consecutive observations
in samples taken at 35, 40 The mean percent
and 45 minutes following the start of the infusion. extractions for all dogs are also given.
Each value is
The mean percent extraction
+ SE of total radioactivity was 31 + 3.9, which was significantly different from zero (Pc.01).
The mean percent extraction of t SE of un-
conjugated estrogens was 27 rt 4.4, and of conjugated estrogens was 16 + 3.7.
Each of these two values was significantly different from zero
(Pc.01). Corresponding values for estrone, estradiol-176, and estrone glucosiduronate
are given
in Table 2.
estrone sulfate
The mean percent
extractions + SE for estrone and for estradiol-176 were 40 + 4.9 and 32 + 2.7, respectively. zero.
Each mean was significantly different from
The mean percent extraction + SE for estrone glucosiduronate was
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460
‘P=ROID=
Table 1. Average concentrations (10-2dpm/ml) of total, unconjugated and conjugated radioactivity in femoral artery (A) and femoral vein (FV) plasma together with the percent extraction [% extr=lOO(l-FV/A)] across the hind limb in 5 dogs following a loading dose and constant infusion of [3H] estrone. Each value is the average concentration in 3 consecutive samples taken at 35, 40 and 45 minutes following the start of the infusion.
Total Dog #
A
15
292
16
Conjugated
Unconjugated
FV
% extr
176
125
29
23
266
223
16
110
40
270
187
31
236
199
16
325
321
1
199
173
13 -
158
152
4
% extr
A
FV
% extr
143
51
131
74
44
522
376
28
91
70
17
639
358
44
184
39
633
512
19
40
381
328
14 -
FV
A
Mean ++ SE ++
31 3.9
27 4.4
16 3.7
P*
c.01
c.01
c.01
++ *
calculated from 3 samples in each of 5 dogs = 15 samples probability
that the mean percent extraction differs from zero
16 + 3.1, which was also significantly different from zero.
In contrast,
the mean extraction of estrone sulfate was negative (-12 + 4.1,) which was significantly different from zero. estradiol-176
glucosiduronate(s)
The mean percent extraction
for
is not given since there was some doubt
as to whether a steady state for this compound was reached in our experiments. The variation with time in the percent extraction values at 35, 40 and 45 minutes was analyzed by regression analysis.
There was no signi-
ficant regression in any case.
However when regression analysis was
applied to the arterial and venous concentrations,
a slow but signifi-
cant increase in both arterial and venous concentrations was observed with time in most instances.
Consequently delay factors associated
with circulation through the hind limb must be considered.
Such delay
factors could affect the calculated percent extraction values (10,12). A correction can be applied if the circulation time is known (10).
Cir-
culation time through the hind limb of the dog is reported to be about 8 sets (11).
Applying the correction as previously described (12) re-
sulted in only trivial changes in the percent extraction values.
Con-
sequently these were ignored. Plots of the ratios of concentration
in thoracic duct lymph to con-
centration in femoral artery plasma versus time for the various radioactive moieties and individual estrogens are shown in Figures l-5.
All
radioactive moieties and individual estrogens measured in arterial plasma were also found in lymph. quite rapid.
Furthermore, entry into the lymph was
Substantial quantities were present within 10 min of the
start of the [3H] estrone infusion. In each case except estrone (Fig. 5), the data were fitted to the curve described by the equation [log (Z/(L/FA x 100)-l) = log X], where (L/FA x 100) is the ratio of concentration in plasma (FA) expressed as a percentage
in lymph (L) to concentration
(and therefore corresponding
to the ordinates in Figs. l-4), X is time in minutes and Z is a selected maximum.
The calculated curves are shown in each of the figures.
value for t (= regression coefficient/SE) all cases, indicating a good fit.
The
varied between 3.1 and 7.5 in
These values for t also indicate
that the regression coefficients differ from zero in each case at the
" *
P*
<:,Ol
4.9 40
c.07
2.7 32
3160
21 -
3150
4280
2270
1910 3590
1389
1125
29
42
4
668
695
13900
0 -
probability that the mean percent extraction differs from zero by chance
c.01
-12 4.1
24800
3490
10330
5280
A
11300
c.01
3.1 16
19 -
4
12
3060 23800
34
9
% extr
6800
4800
FV
Estrone glucosiduronate
-19
-19
-23
% extr
FV
A
Estrone sulfate
calculated from 3 samples in each of 5 dogs= 15 samples
!:"y+++
1980
2510
30 -
20700 14400
40
2040
2880
29
23800 17000
1060
62
1830
39
5600
14800
17
42
360 26
% extr
FV
461
44
606
11030
16
625
A
Estradiol-17g
624
46
3510
6500
15
% extr
FV
A
Dog #
Estrone
Table 2. Average concentration (dpm/ml) of radioactive estrone, estradiol-17B, estrone sulfate and estrone glucosiduronate in femoral artery (A) and femoral vein (FV) plasma, together with the percent extraction [% extr = 100 (l-FV A)] across the hind limb in 5 dogs following a loading dose and constant infusion of [sH] estrone. Each value is the average concentration in 3 consecutive samples taken at 35, 40 and 45 minutes following the start of the infusion.
S 80
0
r
463
TIIROXDS
UNCONJUGkVED
MINUTES
Fig. 1. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for total radjoactivity and for unconjugated radioactivity during an infusion of [ H] estrone Time is in minutes following the start of the infusion. ~!t$~'curve is that of the equation [log (Z/(L/FA x 100)-l) = log i';r where Z is a selected maximum and X is time in minutes.
Pc.01 level. The ratios for total radioactivity and unconjugated radioactivity are shown in Fig. 1.
For total radioactivity the L/FA ratio rose
steadily to reach a plateau at approximately the start of the infusion.
50% at 80-100 min following
The ratio was higher for the unconjugated
estrogens, reaching a plateau of 70% at about 80 min. The L/FA ratio for conjugated estrogens reached a plateau at about 45% at 60-80 min (Fig. 2).
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464
TXIROIDS
60 r
CONJUGA JED
20
I 40
I 60
I
80
I 100
I 120
I 140
MINUTES
Fig. 2. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for conjugated radioactivity during an infusion of C3H] estrone. Time is in minutes following the start of the infusion. The curve is described in Fig. 1.
Corresponding data for the individual conjugates estrone sulfate, estrone glucosiduronate Figs. 3-4.
and estradiol-17B glucosiduronatesare
given in
The L/FA ratio for estrone sulfate reach a plateau at about
45% at 60-80 min (Fig. 3).
Corresponding values for estrone glucosid-
uronate were 45% and 70 min, and for estradiol-17B
glucosiduronate(s)
35% and 90 min (Fig. 4). In contrast, the curve for estrone (Fig. 5) is quite different.
The
early L/FA ratios are higher (about 35%) and decrease to reach a steady state at about 60 min at a value of about 27%. drawn freehand.
The curve in Fig. 5 is
The higher values at the earlier times may reflect the
465
60-
50-
40: x 30e -I
20-
10 -
I
I
I
I
I
I
I
I
20
40
60
80
100
120
140
MINUTES
Fig. 3. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for radioactive estrone sulfate during an infusion of C3H] estrone. Time is minutes following the start of the infusion. The curve is described in Fig. 1.
prior administration
of the loading dose together with delay factors
associated with entry into the lymph. The average flow of lymph through the thoracic duct during these experiment was 0.48 ml per minute.
Average flow of total radioactivity
through the duct was 2.4% of the rate of infusion. DISCUSSION As previously mentioned, a positive percent extraction where A and V are plasma concentrations
([100(1-V/A)],
in artery and representative
efferent vein respectively, may be due to one or more of the following: (1) metabolism within the area drained by the vein, (2) retention in the
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466
TXIROXDB
60 ESTROIVE
GLUCOS~OU~?OIVA~-E
20
I
I
I
I
I
40
60
60
100
120
I 140
MINUTES
Fig. 4. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for radioactive estrone glucosiduronate and estradiol-17f+glucosiduronte(s) during an infusion of [3H] estrone. Time is in minutes following the start of the infusion. The curve is described in Fig. 1.
area unchanged, (3) removal from the area by a route other than the representative metabolism
vein.
Consequently, extraction can be only equated with
if significant contribution by the other two processes can
be excluded.
This in turn can only be accomplished
state) total radioactivity
if (in the steady
in venous plasma is equal to total radio-
activity in arterial plasma (i.e. extraction of total radioactivity
is
zero), since if metabolism were the only process, then radioactive products should be equivalent to radioactive precursors and total radioactivity would remain unchanged.
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TDIR.OXDDII
I 40
I 60
467
50-
IO-
I 20
I 80
1 100
120
I 140
MINUTES
Fig. 5. Relationship with time of the mean lymph (L): femoral artery plasma (FA) concentration ratio (L/FA x 100) for radioactive estrone during an infusion of [3H] estrone. Time is minutes following the start of the infusion. The curve is drawn freehand.
In the present report substantial uptake of total radioactivity by the hind limb of the dog was observed (mean extraction 31%, Table 1). Consequently none of the positive extraction values for unconjugated or conjugated estrogens
(Table 1) or for the various individual estrogens
(Table 2) can be equated solely with metabolism. Thenet uptake (positive extraction) of estrone and estradiol-176 by the hind limb of the dog (Table 2) corresponds with the findings in the human forearm reported by Longcope et al. (5), using similar principles but more sophisticated techniques involving double-label procedures.
Such uptake was equated
with metabolism by these authors, although the extraction of total
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TIIROXDIB
radioactivity by the forearm (if any) was not reported. quantitation of the
metabolism of estrogens
Their
by the forearm
might not be justified if significant extraction of total radioactivity were present. With regard to estrone sulfate, there was evidence for small but significant net formation of this conjugate by the hind limb (mean extraction -12%, Table 2). Longcope et al. reported variable results for estrone sulfate in the human forearm (5).
Some uptake appeared to occur
in two of five experiments, and conversion of estrone to estrone sulfate occurred in one of three experiments. The uptake of estrone glucosiduronate
(mean extraction 16%, Table 2)
by the hind limb of the dog is in contrast to the net release of estrone sulfate. workers
Estrone glucosiduronate was not studied by Longcope and co(5).
For the reasons given above, the interpretation of the uptake of estrone, estradiol-176 and estrone glucosiduronate the dog noted in these experiments
is difficult.
by the hind limb of Some metabolism would
be expected on general grounds and from previously published -___ in vivo work on the metabolism of estrogens and androgens by muscle and fat (5-8) in various species.
Our present evidence for the net formation of
estrone sulfate and the evidence from the work of Longcope et al. for the interconversion of estrone and estradiol-176 (5) would support this view.
in the human forearm
On the other hand, our finding of substan-
tial uptake of total radioactivity by the hind limb must indicate either some retention unchanged in the area, or some removal by a route other than the femoral vein, or both. Retention seems unlikely.
The loading dose would be expected to
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TPROXD= The extraction of
occupy rapidly most available storage sites.
total radioactivity did not change substantially between 10 and 45 min It would seem unlikely that storage would con-
(60 min in two dogs).
tinue at the same rate over this period of time. take up estrogens preferentially
Adipose tissue may
(14, 15) but there are no extensive
deposits of fat in the dog hind limb (in contrast to the splanchnic area, for instance).
However, it is clear that retention cannot be
completely excluded. The alternative explanation, namely, removal by some route other than the vein, appears more likely. be the lymphatics.
The only such route would appear to
The data in Figs. 1-5 and Table 3 indicate that
estrogens do in fact enter the lymphatics promptly and in substantial concentration after infusion of [3H] estrone.
So far as the authors
are aware, this is the first published study of estrogens in lymph in any species. Whether the entry of estrogens into lymph can account for the extraction of total radioactivity found across the hind limb in the present study in not yet clear.
Thoracic duct lymph,studied here, drains much
of the body and the concentrations
found in the duct may be higher or
lower than those in the lymphatics draining the hind limb. the quantitative
Furthermore,
importance of the lymphatic route will depend upon
lymph flow through the hind limb as well as lymph concentration. some explanation
However
is needed for the substantial extraction of total radio-
activity by the hind limb.
Entry into the lymphatics appears to be the
best explanation at the present time.
470
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TIlROIDb
ACKNOWLEDGEMENTS Dr. D.C. Collins is the recipient of USPHS Career Research Development Award 5-K04-AM-70381 from the National Institute of Arthritis, Metabolism and Digestive Diseases. This research was supported by NIH Grant 5-ROl-AM-02221 and 5-ROl-AM-13468. The able technical assistance of Hugh Robinson, Robinette King and Roy Weems is gratefully acknowledged. REFERENCES 1. Address reprint requests to Dr. D.C. Collins, Department of Medicine, Emory University School of Medicine, 69 Butler St., S.E., Atlanta, Georgia 30303. 2. The following trivial names and abbreviations for steroids have been used. estrone (E )=3-hydroxyestra-1,3,5(10)-trien-17-one; estradiol- 1 7B(E2)=estra-l,3,5(lO)-triene-3,17@-diol; estrone glucosiduronate (ElG)=17-oxoestra-1,3,5(10)-trien-3-yl-B-Dglucopyranosiduronate; estradio1-1713-g1ucosiduronate-17B-hydroxyestra-1,3,5(10)-trien-3-y1R-D-glucopyranosiduronate and 3-hydroxyestra-1,3,5(10)-trien-17Byl-P-D-glucopyranosiduronate; estrone sulfate (ElS)=l7-oxoestra-1,3,5(10)-trien-3-yl-sulfate. J. CLIN. INVEST. -47:93, 3. Longcope, C., Layne, C.S. and Tait, J.F., (1968). 4. Collins, D.C., Robinson, H.D., Howard, C.M. and Preedy, J.R.K., J. CLIN. INVEST. 49:2326 (1970). 5. Longcope, C., Pratt, J.H., Schneider, S.H. and Fineberg, S.E., J. CLIN. ENDOCRINOL. METAB. 43:1134 (1976). 6. Chapdelaine, A.. J. CLIN.INVEST. 48:2063 (1969). A., REV. CAN. BIOL. 31:135 7. Bleau, G., Roberts, K.D. and Chapdsaine, (1972). Schneider, S.H. and Fineberg, S.E., 8. Longcope, C., Pratt, J.H., STEROIDS 28:521 (1976). 9. Collins, KC., STEROIDS 18:341 (1971). 10. Zierler, K.L., J. CLIN. NVEST. 36:2111 (1961). AND CIRCULATION. 11. Altman. P.L. and Dittmer, D.C., FSPIRATION Federation of American Societies of Experimental Biology, Bethesda, Md. 1971, p. 414. 12. Collins, D.C., Balikian, H.M. and Preedy, J.R.K., ENDOCRINOL. 96: 1543 (1975). 13. Collins, D.C., Balikian, H.M. and Preedy, J.R.K., ENDOCRINOL. 99:420 (1976). 14. Balikian, H., Southerland, J., Howard, C.M. and Preedy, J.R.K., ENDOCRINOL. 82:500 (1964). 15. Bleau, G.. Roberts, K.D. and Chapdelaine, A., J. CLIN. ENDOCRINOL. METAB. 39:236 (1976).