ALTERATIONS IN SULFOBROMOPHTHALEIN SODIUM REMOVAL MECHANISMS FROM BLOOD DURING NORMAL PREGNANCY

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ALTERATIONS IN SULFOBROMOPHTHALEIN

SODIUM-REMOVAL MECHANISMS FROM

BLOOD DURING NORMAL PREGNANCY

Burton Combes, … , Billie D. Mitchell, Victor Trammell

J Clin Invest. 1963;42(9):1431-1442. https://doi.org/10.1172/JCI104828.

Research Article

Find the latest version:

http://jci.me/104828/pdf

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Journal ofClinical Investigation Vol. 42, No. 9, 1963

ALTERATIONS IN

SULFOBROMOPHTHALEIN

SODIUM-REMOVAL

MECHANISMS FROM BLOOD DURING NORMAL PREGNANCY*

By BURTON COMBES,t HISAO SHIBATAJREUBEN ADAMS, BILLIE D. MITCHELL, AND VICTOR TRAMMELL

(From the Departments of Intcrnal Medicine and Obstetrics and Gynecology, Univcrsity of Texas, Souithwestern Medical School, Dallas, Tex.)

(Submitted for publication March 21, 1963; accepted May 23, 1963)

During the past few years, we have been im-pressed by the observation that our patients with pre-eclampsia and eclampsia usually demonstrate sulfobromophthalein sodium (BSP) retention in blood in the 45-minute BSP test. On the basis of these findings, we undertook a study of hepatic BSP-removal mechanisms during the course of normal pregnancy. Increased BSP retention in 45 minutes was not detected in normal pregnancy when compared to results obtained in a group of nonpregnant women ofcomparable age. When hepatic BSP-removal mechanisms were appraised quantitatively by a prolonged-infusion method (1, 2), however, significant changes in both hepatic

uptake and excretion of BSPwere observed. The following study presents in detail such data ob-tained from women in various stages of normal pregnancy and in the early postpartum period,

and compares these findings with data obtained in

a separate group of control nonpregnant women.

METHODS

The method used in the present studies was devisedby Wheeler and his associates (1, 2) and is based on the observations that removal of BSP from plasma depends

on the simultaneous operation of at least two separate

hepatic mechanisms: 1) uptake of BSP into a hepatic

*_This_work_was _{supported by} _{research grants from the}

U. S. Public Health Service (H-3439, A-6082), and frcm Parke, Davis & Co. Part of this work has appeared in abstract form (Clin. Res. 1962, 10, 189; 1963, 11, 58)

and was presented at the Annual Meeting of the Ameri-canAssociation for the Studyof Liver Diseases, Chicago, Ill., November, 1962, and at the Annual Meeting of The

Southern Society for Clinical Research, New Orleans,

La., January, 1963.

t Part of this work was performed during the tenure ofan Established Investigatorship of theAmerican Heart Association. Recipient of Research Career Development

Award of the U. S. Public Health Service.

tRecipient of Postdoctoral training fellowship from Parke, Davis & Co., 1961-1962. Present address: School of Medicine, Keio University, Tokyo, Japan.

"storage compartment," and 2) active secretion into bile (1-8). The quantity of BSP taken up into the "storage

compartment" appears to be directly proportional to plasma concentration (1). Thus, uptake into storage during a period of changing plasma concentration can be represented in symbols as S X AP/At, where the "rela-tive storage capacity" S, a proportional factor, is de-fined as the number of milligrams of BSP stored in the liver per milligram per 100 ml of plasma concentration,

and AP/At signifies the rate of change of plasma con-centration in milligrams per 100 ml per minute. Se-cretion of BSP into bile is a rate-limited process

char-acterized by a maximal excretory rate or Tm (1, 2, 8), defined intermsof milligrams per minute. The maximal rate of BSP secretion into bile is achieved when plasma BSP concentration is maintained in excess of 2 to 3 mg per 100 ml during prolonged infusions of BSP (1, 8). At these plasma concentrations, the general equation for the hepatic removal rate of BSP (1), RH, in milligrams per minute, is as follows: RH=SX (AP/At) +Tm.

Values for RH and AP/At obtainedduring two or more different rates of BSP infusion are substituted in the formula, providing a set of simultaneous equations that canbe solved for both S and Tm. In practice, values for RH and AP/At were obtained during three separate

in-fusion rates of BSP, each given for an hour. As indi-cated by Wheeler, when the corresponding values for RH and AP/At are plotted on the ordinate and abscissa,

respectively, the relationship between RH and AP/At ap-pears to be approximately linear. Thus, the equation of the line that best fits the three points is calculated by the method of least squares (9). Since the general equation for hepatic removal rate of BSP is in the form of a

straight line, y=mX + c, S is equal to the slope of the

least-squares line, and Tm is equal to the value of the y intercept.

Procedures

Single, prolonged-infusion studies were conducted be-fore surgery on 10 control nonpregnant women in the

reproductive age range admitted to the gynecology serv-ice, usually for therapy for stress incontinence or for simple hysterectomy; on 15 women in various stages of normal pregnancy; and on 11 women during the first week postpartum. The studies were usually carried out

in the morning, approximately 1 hour after a fat-free breakfast. The procedure used was essentially the same

as thatdescribed by Wheeler, Meltzer, and Bradley (2),

with the exception that the BSP administered was

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HEPATIC BSP REMOVAL MECHANISMS IN PREGNANCY

luted in isotonic saline. Plasma volume was measured

in each subject during the first hour of the study with

I"~-labeled human serum albumin. A plasma sample was

obtained 10 minutes after injection of approximately 5 uc

ofradioactivetracerfor estimation of plasma volume. A 45-minute BSP test (10) was carried out in most

con-trol and pregnant subjectsonthe day before, and in

post-partumwomen, just before the prolonged-infusion study. In 14additional studies, constantinfusions of BSP were

administered for 24 to 156 minutes to women in labor.

At the time of delivery, samples of maternal and fetal cordbloodwereobtained simultaneously for estimation of

plasma concentration of BSP. Placentas were obtained

from10 of these womenand analyzed for BSP content. The quantity of BSP excreted into urine during 65 to 97minutes of BSP infusionwas determined in four other

studies conducted in women at term. Analytical methods

Plasma. One ml of each plasma sample was mixed

with 9 ml of 0.1 N KOH. The optical density was

de-termined ina Beckman DU spectrophotometer set at 580

mit. Plasma obtained before infusion of BSP wastreated

similarly and served as a blank. Standard BSP curves

were prepared for each patient by addition of known

amounts of BSP to plasma obtained before BSP

infu-sion. Since blank samples of fetal cord blood could not

be obtained, BSP concentration in fetal cord plasma was

estimated by the method of Gaebler (11).

Infuision mixture and urine. Samples of infusion

mix-ture and urine samples were diluted with distilled water

and alkalized, and the optical density was determined

at 580 m1A. For analyses of urine, samples obtained just

before infusion of BSP were diluted to the same extent

as the sample containing BSP and served as blanks.

Chromatography of BSP compoundsin plasma. Equal volumes ofplasma from each of the four plasma samples obtained during each hour of BSP infusion were pooled.

BSP compounds were extracted from the pooled plasma

and prepared for paper chromatography on Whatman

3MM paper asoutlined by Carbone, Grodsky, and Hjelte

(12). The chromatograms were developed in a

descend-ing system employing glacial acetic acid:water:n-propyl alcohol (1: 5: 10 by vol), for 16 to 18 hours. BSP bands were identified by exposing the dried papers to ammonia vapors. BSP was eluted from each band and

the concentration determined by methods described in detail in previous publications (13, 14). One or two

bands corresponding to BSP conjugates anda band

con-taining free BSP were identified on the chromatograms.

The proportion of BSP appearing as conjugated BSP was determined by dividing the sum of the optical

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IN MAXIMAL EXCRETORY RATE OF BSP INTO BILE (BSP Tm) DURING NORMAL PREGNANCY

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FIG. 2. ALTERATIONS IN HEPATICRELATIVE STORAGE CAPACITY FOR BSP (S) DURING NORAMAL PREGNANCY AND THE FIRST WEEK POSTPARTUM.

sities of the conjugate bands by the sum of the optical densities of the conjugate and free BSP bands. For these studies, it was assumed that the molecular

ex-tinction coefficient for free and conjugated BSP was

identical.

Extraction of BSP from placenta. The amnion and chorion were stripped off, and the umbilical cord was

cut at its juncture with the placenta. The placenta was

weighed, cut into small pieces roughly 1-inch square, and

homogenized in a Waring Blendor with a volume of

dis-tilledwater equal to the weightof the placenta. Two-ml samples of homogenate, equivalent to 1 g of placenta,

were pipetted in triplicate into glass-stoppered, conical

centrifuge tubes. To extract BSP, 4ml of 80% acetone

was added to each tube. After thorough shaking, the

tubes werecentrifugedat 4,500rpm for 10minutes. The supernatant fluid was removed and the precipitate

ex-tractedtwice with 3 ml of80% acetone. The supernatant fluids were pooled and the volume recorded. Duplicate

3-ml samples ofthe combined acetone extractwere mixed

with 1 ml of 0.1 N KOH, and the optical density was

determined by using 0.1 N KOH as a blank in a Beck-man DU spectrophotometer set first at 580 mu and then

at 620 m/A. The alkalinized acetone extracts exhibited varying amounts of turbidity. The optical density at

580 myAof similarly turbid alkalinized acetone extracts of placentas free of BSP averaged 1.10 times the optical density at 620 my. Thus, the BSP content of each

pla-centa was calculated as follows: [OD 580-1.10 OD 620

(unknown) ]/ [OD 580-1.10 OD 620 (standard)] X

con-centration of standard solution in milligrams per 100 ml

Xvolume of acetone extract in fractions of 100 mlX

weight of the placenta in grams. Since recovery of

known amounts of BSP homogenized with placentas averaged 62%, the valueabove was multiplied by 100/62.

RESULTS

1. Maximal excretory rate of BSP into bile,

BSP Tic. In 10 control nonpregnant women, the

average BSP

T.

was 8.5 mg per minute 1.3

SD. The individual results are listed in Table I.

T.,

appeared to rise with increasing surface area,

but the correlation coefficient of 0.5 was not

sta-tistically significant, p > 0.05. BSP Tm

de-creased duringthe latter partof normal pregnancy

(Table II, Figure 1). The average

T,,,

in the

last half ofpregnancy of 6.2 mg per minute 1.7

SD represented a fall of

277o

below control, a

statistically significant decrease, t= 3.38, p<

0.01. A rapid return of Tm to control values

was observed during the first week postpartum

(Table III, Figure 1).

2. Hepatic relative storage capacity, S. The

average value for S in 10 control nonpregnant

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women was 43.4 mg per mg per 100 ml + 12.3 SD (Table I). No correlation between S and surface area was apparent. S did not change during the early part of pregnancy (Table II, Figure 2). Later, however, S rose markedly and averaged 96.5 mg per mg per 100 ml ± 22.6 SD during the last half of pregnancy. The

in-crease of

122%

above control in the latter half

of pregnancy was highly significant, t = 6.56, p < 0.001. After delivery, S tended to fall toward control values, although it still remained elevated as long as one week postpartum in some patients

(Table III, Figure 2).

3. BSP concentration in maternal and fetal cord plasma. Constant infusions of BSP were administered to 14 women at term for a mean

period of 73 minutes. During this time, an

aver-age of 1,177 mg of dye was removed from the circulation. The concentration of BSP in

ma-ternal plasma at the time of delivery averaged 9.2 mg per 100 ml. In fetal cord plasma, sampled

at the same time, no BSP was detected in 10 specimens. BSP was found in the other 4. In these instances, the concentration of dye, in

milli-grams per 100 milliliters, in fetal cord and

ma-ternal plasma, respectively, was 0.08 fetal to 14.6 maternal; 0.16 to 5.8; 0.63 to 17.0; and3.6 to7.6. 4. BSP uptake by placenta. BSP was infused foran average of 70 minutes in 10women at term.

Of the 1,085 mg of BSP removed from blood

during this period, an average of only 7.2 mg could be accounted for in the placentas. Since placentas contain approximately 50 ml of

ma-ternal plasma (15), the quantity of BSP to be expected in this volume of plasma (maternal

plasma concentration of BSP in milligrams per 100 ml at time of delivery x

0.5)

averaged 5.1 mg. Actual tissue content of BSP was

approxi-mately 2.1 mg of BSP per placenta, therefore. 5. Urinary excretion of BSP. An average of

1,526 mg of BSP was removed from blood dur-ing a mean infusion period of 83 minutes in four

women at term. Only 8.1 mg of BSP was

ex-creted into urine during this period, on the average.

DISCUSSION

Almost all of the results of the 45-minute BSP

testscarried outduring thecourseof normal

preg-nancy (Table II) fell within the range of values

obtained in a group of control nonpregnant

women. It seemed unlikely, therefore, that any

alterations in hepatic BSP-removal mechanisms

would be noted (luring the course of normal

preg-nancy. However, two significant changes were

observed when the prolonged-infusion method

of Wheeler and his associates (1, 2) was utilized. Thus, S increased markedly, whereas BSP Tm,

decreased during the last half ofpregnancy. It is

apparent that the Wheeler method provides an extremely useful tool for examining hepatic BSP-removal mechanisms. It not only permits an appraisal of both hepatic uptake and biliary

ex-cretion of BSP in quantitative terms, but

ob-viously it is capable of detecting significant changes even when the sensitive clinical BSP test yields normal results.

The most impressive finding in the present studies was the marked rise in S during the last half of pregnancy. There are several possible explanations for this change. First, it was neces-sary to demonstrate that significant BSP removal from blood in sites other than the liver did not

account spuriously for the higher values of S

observed in the last half of pregnancy. Since S was as high at 24 weeks of gestation as at 40, it seemed unlikely that BSP uptake by the placenta

or the fetus, which would enlarge considerably during this period of gestation, contributed in

any significant way to BSP removal from plasma. This was corroborated byour studies which dem-onstrated that virtually no BSP is taken up by or

crosses the placenta. Others have examined

transplacental movement of BSP after adminis-tration of a single iv injection of 5 mg per kg.

Freiheit detected no dye in cord blood of two

infants delivered within half an hour of BSP administration (16). Smith, Moya, and Shnider

(17) observed BSP in umbilical vein blood as

early as 20 seconds after BSP injection, and low levels of BSP were found in nine of 15 subjects.

No BSP was detected in umbilical vein blood of

six infants. In our constant infusion studies in which cord blood was sampled, no measurable amount of BSP was found in 10 of 14 subjects.

If a large quantity of BSP had passed into the

fetal circulation, it seems likely that relatively

high levels of BSP would have been detected,

(10)

since BSP removal from blood is delayed in thenewborn infant (18-22). In the present stud-ies, urinary loss of BSP was small in pregnancy. On the basis of earlier observations (1, 23-25), it seems unlikely that other extrahepatic sites accounted for a marked increase in BSP removal from blood, although this possibility is not ex-cluded. Significant uptake of BSP by tissues other than the liver apparently does not explain the rise in S.

Before attributing increased S to an intrinsic change in the liver, it is necessary to consider other extrahepatic factors that might result in increased hepatic BSP storage in pregnancy. With the method used in these studies, it is as-sumed that the quantity of BSP taken up into storage for each increment in plasma concentra-tion of 1 mg per 100 ml is the same over a limited range of plasma concentration (1). It is conceivable that the capacity of the storage mecha-nism differs when changes in plasma concentra-tion occur at low and high plasma levels of BSP. If the mechanism accounting for storage is com-posed of units with different affinities for BSP, components withhigh affinity would take up BSP first, thus at low plasma levels. Units with progressively lower affinity would remain as the quantity of BSP stored increases during rising plasma concentration. Thus lower values for S would be expected at higher plasma levels. In the present studies, average plasma levels of BSP during each hour of BSP infusion were somewhat lower in women in the last half of pregnancy than in control nonpregnant women

(5.53, 4.18, and 7.27 mg per 100 ml in pregnant women compared to 8.95, 7.01, and 8.88 mg per 100 ml in control nonpregnant women, Tables I and II). Apparently, in the dog, S is relatively

constant over a range of BSP levels in plasma of

at least 3 to 12 mg per 100 ml (26). The

plasma concentrations attained in the present studies fell well within this range, and if S be-haves in the human as it does in the dog, it seems unlikely that the small differences in plasma levels could account for the large changes in S observed during pregnancy.

The degreeof

protein-binding

ofBSP inplasma

may exert

aIn

influence on

_uptake

of BSP into

storage. It is possible that the quantity of BSP

stored in the liver is proportional to a small

frac-tion of unbound dye in plasma rather than to

total plasma concentration of BSP. Thus,

in-creased unbound BSP in plasma of pregnant women might result in increased S. In prelimi-nary studies (27), ultrafiltrates of plasma,

ob-tained from both control and pregnant women,

to which BSP had been added in vitro revealed nofree BSP inplasma water with total BSP

con-centrations in plasma of up to 20 mg per 100 ml.

These observations suggest, therefore, that

differ-ences in protein-binding over the range of plasma BSP concentrations attained in the present

stud-ies did not influence values of S.

A rise in S might be considered to result from a decrease in Tm. This seems unlikely,

how-ever, for in at least three situations-in patients with the Dubin-Johnson syndrome (2), after

acute ligation of the common bile duct in dogs (26), and during administration of

17a-ethyl-19-nortestosterone (37)-S has remained normal

when Tm, is decreased markedly.

It seems reasonable to conclude, therefore, that an increase in S during the last half of pregnancy indicates that the relative storage capacity of

the liver for BSP increases, and furthermore, that

this alteration is due to an intrinsic change in the

liver. Such an increase in S could be accounted

for by an increase in hepatic mass, by increased

storage capacity of each unit of liver

tissue,

or

by

both. As for increased hepatic mass in normal

pregnancy in the human, few data are available

for analysis. A review of autopsy information

derived from women dying after a brief illness

during pregnancy in our hospital revealed an average liver weight of approximately 1,700 g, with no significant difference in liver mass

dur-ing early and late pregnancy. Thus, an increase

in size of the liver does not explain the rise in S.

Normally, BSP is concentrated in human liver

three to four times above plasma concentration,

on the average (2).

Although

the nature of the

concentrating mechanism is not known-whether

it is a liver protein with greater affinity for BSP

than plasma protein, an active transport

mecha-nism, or something else-, an increase in capacity

of this mechanism in each unit of liver tissue is

the most likely explanation for the rise in S

during pregnancy.

(11)

The implication of this observation appears to extend beyond hepatic uptake of BSP. Ob-viously, the human liver is not usually exposed to BSP. A rise in storage capacity for this dye could hardly be considered a specific response, therefore. It seems reasonable to expect that in-creased hepatic storage capacity for a variety of compounds, including drugs, will be found during pregnancy.

The fall in Tm in the last half of pregnancy may also be accounted for by several possibilities. First, if the liver holds on to BSP more avidly, less BSP may be available for excretion. The observation that S remained increased at a time when Tm was normal in several postpartum pa-tients suggests another mechanism, however.

Second, interference with BSP conjugation might result in decreased Tm. Previous work indicated that conjugation of BSP with glutathione

(28-30), a process catalyzed by a liver enzyme (31), does not influence the extent to which BSP is

stored in the liver (32). BSP Tm is dependent

on conjugation, however, since conjugated BSP is delivered into bile at a faster rate than free BSP (32). The components of the

BSP-gluta-thione conjugating system have not been

ex-amined in human liver during pregnancy. In the rat, a decrease in BSP conjugating enzyme

activity with normal levels of glutathione has been found during the last 5 days of gestation (14). When liver glutathione levels have been maintained, however, a moderate decrease in en-zyme activity has not resulted in a decrease in BSP Tm in the rat (32). Whether conjugation

is impaired as a result of a critical decrease in enzyme activity or glutathione content in the human liver must await further studies. An analysis of BSP compounds appearing in plasma

was carried out in order to detect any major changes in conjugation that might occur in the present studies. BSP conjugates were observed in plasma during the prolonged infusions of BSP (Tables I toIII). Theproportionof totalplasma

BSP accounted for by BSP conjugates during

each hour of infusion was almost the same in patients during the last half of pregnancy and in control nonpregnant women (5.8, 21.6, and 20.9%

inpregnancy, as comparedto6.4, 26.4, and 24.4%o

in controls). The smalldifferences between

preg-nant and control patients in the averages for each hour were not significant statistically. Since the quantity of BSP infused was approximately the same in pregnant and nonpregnant subjects,

the data above suggest that major changes in BSP conjugation in pregnancy did not affect Tm.

Finally, decreased Tm may result from an im-pairment of the transport processes involved in the delivery of both conjugated and free BSP from liver cells to bile. In this regard, it is of interest that a syndrome of obstructive jaundice has been observed during the latter part of pregnancy (33-36). After delivery, the obstruc-tive features disappear, and hepatic function

re-turns to normal. Obstructive jaundice may recur

with subsequent pregnancies. The nature of the intrahepatic disturbance appears to involve im-pairment of biliary secretory mechanisms. It is tempting to speculate that the decrease in BSP Tm observed in the present studies reflects a

similar response of the liver, although obviously a

less marked one, to changes occurring normally during pregnancy. According to this view, ob-structive jaundice might result either from an

increase in quantity of the factor thataccounts for decreased BSP Tm in normal pregnancy, or from

a greater sensitivity of biliary secretory mecha-nisms to normal amounts of this factor found in pregnancy.

Analysis of various mechanisms that might

account for both the rise in S and the fall in Tm in pregnancy appears to indicate that altera-tions in two independent

hepatic

systems are

involved. Furthermore, since the

changes

in hepatic BSP-removal mechanisms occur during

the last half of pregnancy, and then return to

or toward normal in the first week after

delivery,

it seems likely that they are mediated by in-creased hormone levels in pregnancy,

probably

of estrogens, progesterone, or both. With

de-crease in hormone levels after

delivery,

BSP-removal mechanisms return toward normal.

SUMMARY

Hepatic sulfobromophthalein sodium

(BSP)-removal mechanisms have been appraised

(12)

HEPATIC BSP REMOVAL MECHANISMS IN PREGNANCY pacity for BSP, S, defined as the number of

milligrams of dye taken up into storage for each

increment of plasma concentration of 1 mg per

100 ml and 2) the maximal excretory rate of

BSP into bile, BSP Tm, inmilligrams per minute.

S rose

122%

during the last half of pregnancy,

then returned to or toward normal during the

first week postpartum. In contrast, BSP Tm

decreased 27% in the last halfof pregnancy, then

rapidly increased to normal levels after delivery. These changes appear to reflect alterations in two independent hepatic mechanisms.

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