ALTERATIONS IN SULFOBROMOPHTHALEINSODIUM-REMOVAL MECHANISMS FROMBLOOD 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

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Journal of Clinical InvestigationVol. 42, No. 9, 1963

ALTERATIONS IN SULFOBROMOPHTHALEINSODIUM-REMOVALMECHANISMSFROMBLOODDURINGNORMALPREGNANCY*

By BURTONCOMBES,t HISAO SHIBATAJ REUBENADAMS, BILLIE D. MITCHELL, ANDVICTOR TRAMMELL

(From the Departments of Intcrnal Medicine and Obstetrics and Gynecology, Univcrsity ofTexas, 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 withpre-eclampsia and eclampsia usually demonstratesulfobromophthalein sodium (BSP) retention inblood in the 45-minute BSP test. On the basisof these findings, we undertook a study of hepaticBSP-removal mechanisms during the course ofnormal pregnancy. Increased BSP retention in45 minutes was not detected in normal pregnancywhen compared to results obtained in a groupof nonpregnant women of comparable age. Whenhepatic BSP-removal mechanisms were appraisedquantitatively by a prolonged-infusion method (1,2), however, significant changes in both hepaticuptake and excretion of BSP were observed. Thefollowing study presents in detail such data ob-tained from women in various stages of normalpregnancy and in the early postpartum period,and compares these findings with data obtained ina separate group of control nonpregnant women.

METHODS

The method used in the present studies was devised byWheeler and his associates (1, 2) and is based on theobservations that removal of BSP from plasma dependson the simultaneous operation of at least two separatehepatic mechanisms: 1) uptake of BSP into a hepatic

* This work was supported by research grants from theU. S. Public Health Service (H-3439, A-6082), and frcmParke, Davis & Co. Part of this work has appeared inabstract form (Clin. Res. 1962, 10, 189; 1963, 11, 58)and was presented at the Annual Meeting of the Ameri-can Association for the Study of Liver Diseases, Chicago,Ill., November, 1962, and at the Annual Meeting of TheSouthern Society for Clinical Research, New Orleans,La., January, 1963.

t Part of this work was performed during the tenureof an Established Investigatorship of the American HeartAssociation. Recipient of Research Career DevelopmentAward of the U. S. Public Health Service.

t Recipient of Postdoctoral training fellowship fromParke, Davis & Co., 1961-1962. Present address: Schoolof Medicine, Keio University, Tokyo, Japan.

"storage compartment," and 2) active secretion into bile(1-8). The quantity of BSP taken up into the "storagecompartment" appears to be directly proportional toplasma concentration (1). Thus, uptake into storageduring a period of changing plasma concentration can berepresented 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 theliver 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 in terms of milligrams per minute. The maximalrate of BSP secretion into bile is achieved when plasmaBSP concentration is maintained in excess of 2 to 3 mgper 100 ml during prolonged infusions of BSP (1, 8).At these plasma concentrations, the general equation forthe hepatic removal rate of BSP (1), RH, in milligramsper minute, is as follows: RH= S X (AP/At) + Tm.

Values for RH and AP/At obtained during two or moredifferent rates of BSP infusion are substituted in theformula, providing a set of simultaneous equations thatcan be solved for both S and Tm. In practice, values forRH 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 forRH 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 ofthe line that best fits the three points is calculated by themethod of least squares (9). Since the general equationfor hepatic removal rate of BSP is in the form of astraight line, y = mX + c, S is equal to the slope of theleast-squares line, and Tm is equal to the value of the yintercept.

Procedures

Single, prolonged-infusion studies were conducted be-fore surgery on 10 control nonpregnant women in thereproductive age range admitted to the gynecology serv-ice, usually for therapy for stress incontinence or forsimple hysterectomy; on 15 women in various stages ofnormal pregnancy; and on 11 women during the firstweek postpartum. The studies were usually carried outin the morning, approximately 1 hour after a fat-freebreakfast. The procedure used was essentially the sameas that described by Wheeler, Meltzer, and Bradley (2),with the exception that the BSP administered was di-

1431

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luted in isotonic saline. Plasma volume was measuredin each subject during the first hour of the study withI"~-labeled human serum albumin. A plasma sample was

obtained 10 minutes after injection of approximately 5 uc

of radioactive tracer for estimation of plasma volume. A45-minute BSP test (10) was carried out in most con-

trol and pregnant subjects on the day before, and in post-partum women, just before the prolonged-infusion study.

In 14 additional studies, constant infusions of BSP were

administered for 24 to 156 minutes to women in labor.At the time of delivery, samples of maternal and fetalcord blood were obtained simultaneously for estimation ofplasma concentration of BSP. Placentas were obtainedfrom 10 of these women and analyzed for BSP content.

The quantity of BSP excreted into urine during 65 to97 minutes of BSP infusion was determined in four otherstudies conducted in women at term.

Analytical methods

Plasma. One ml of each plasma sample was mixedwith 9 ml of 0.1 N KOH. The optical density was de-termined in a Beckman DU spectrophotometer set at 580mit. Plasma obtained before infusion of BSP was treatedsimilarly and served as a blank. Standard BSP curves

were prepared for each patient by addition of knownamounts of BSP to plasma obtained before BSP infu-

sion. Since blank samples of fetal cord blood could notbe 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 waterand alkalized, and the optical density was determinedat 580 m1A. For analyses of urine, samples obtained justbefore infusion of BSP were diluted to the same extentas the sample containing BSP and served as blanks.

Chromatography of BSP compounds in plasma. Equalvolumes of plasma from each of the four plasma samplesobtained during each hour of BSP infusion were pooled.BSP compounds were extracted from the pooled plasmaand prepared for paper chromatography on Whatman3MMpaper as outlined by Carbone, Grodsky, and Hjelte(12). The chromatograms were developed in a descend-ing system employing glacial acetic acid: water: n-propylalcohol (1: 5: 10 by vol), for 16 to 18 hours. BSPbands were identified by exposing the dried papers toammonia vapors. BSP was eluted from each band andthe concentration determined by methods described indetail in previous publications (13, 14). One or twobands corresponding to BSP conjugates and a band con-

taining free BSP were identified on the chromatograms.The proportion of BSP appearing as conjugated BSPwas determined by dividing the sum of the optical den-

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sities of the conjugate bands by the sum of the opticaldensities of the conjugate and free BSP bands. Forthese 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 wascut at its juncture with the placenta. The placenta was

weighed, cut into small pieces roughly 1-inch square, andhomogenized in a Waring Blendor with a volume of dis-tilled water equal to the weight of the placenta. Two-mlsamples of homogenate, equivalent to 1 g of placenta,were pipetted in triplicate into glass-stoppered, conicalcentrifuge tubes. To extract BSP, 4 ml of 80% acetonewas added to each tube. After thorough shaking, thetubes were centrifuged at 4,500 rpm for 10 minutes. Thesupernatant fluid was removed and the precipitate ex-

tracted twice with 3 ml of 80% acetone. The supernatantfluids were pooled and the volume recorded. Duplicate3-ml samples of the combined acetone extract were mixedwith 1 ml of 0.1 N KOH, and the optical density was

determined by using 0.1 N KOHas a blank in a Beck-man DU spectrophotometer set first at 580 mu and thenat 620 m/A. The alkalinized acetone extracts exhibitedvarying amounts of turbidity. The optical density at580 myA of similarly turbid alkalinized acetone extracts ofplacentas free of BSP averaged 1.10 times the opticaldensity 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 OD620 (standard) ] X con-

centration of standard solution in milligrams per 100 mlX volume of acetone extract in fractions of 100 ml Xweight of the placenta in grams. Since recovery ofknown amounts of BSP homogenized with placentasaveraged 62%, the value above was multiplied by 100/62.

RESULTS

1. Maximal excretory rate of BSP into bile,BSP Tic. In 10 control nonpregnant women, theaverage BSP T. was 8.5 mg per minute 1.3SD. 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 during the latter part of normal pregnancy

(Table II, Figure 1). The average T,,, in thelast half of pregnancy of 6.2 mg per minute 1.7SD represented a fall of 277o below control, a

statistically significant decrease, t = 3.38, p <0.01. A rapid return of Tm to control valueswas observed during the first week postpartum(Table III, Figure 1).

2. Hepatic relative storage capacity, S. Theaverage value for S in 10 control nonpregnant

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women was 43.4 mg per mg per 100 ml + 12.3SD (Table I). No correlation between S andsurface area was apparent. S did not changeduring the early part of pregnancy (Table II,Figure 2). Later, however, S rose markedlyand averaged 96.5 mg per mg per 100 ml ± 22.6SD during the last half of pregnancy. The in-crease of 122% above control in the latter halfof pregnancy was highly significant, t = 6.56,p < 0.001. After delivery, S tended to fall towardcontrol values, although it still remained elevatedas long as one week postpartum in some patients(Table III, Figure 2).

3. BSP concentration in maternal and fetalcord plasma. Constant infusions of BSP wereadministered to 14 women at term for a meanperiod of 73 minutes. During this time, an aver-age of 1,177 mg of dye was removed from thecirculation. The concentration of BSP in ma-ternal plasma at the time of delivery averaged 9.2mg per 100 ml. In fetal cord plasma, sampledat the same time, no BSP was detected in 10specimens. BSP was found in the other 4. Inthese instances, the concentration of dye, in milli-grams per 100 milliliters, in fetal cord and ma-ternal plasma, respectively, was 0.08 fetal to 14.6maternal; 0.16 to 5.8; 0.63 to 17.0; and 3.6 to 7.6.

4. BSP uptake by placenta. BSP was infusedfor an average of 70 minutes in 10 women at term.Of the 1,085 mg of BSP removed from bloodduring this period, an average of only 7.2 mgcould be accounted for in the placentas. Sinceplacentas contain approximately 50 ml of ma-ternal plasma (15), the quantity of BSP to beexpected in this volume of plasma (maternalplasma concentration of BSP in milligrams per100 ml at time of delivery x 0.5) averaged 5.1mg. Actual tissue content of BSP was approxi-mately 2.1 mg of BSP per placenta, therefore.

5. Urinary excretion of BSP. An average of1,526 mg of BSP was removed from blood dur-ing a mean infusion period of 83 minutes in fourwomen at term. Only 8.1 mg of BSP was ex-creted into urine during this period, on theaverage.

DISCUSSION

Almost all of the results of the 45-minute BSPtests carried out during the course of normal preg-

nancy (Table II) fell within the range of valuesobtained in a group of control nonpregnantwomen. It seemed unlikely, therefore, that anyalterations in hepatic BSP-removal mechanismswould be noted (luring the course of normal preg-nancy. However, two significant changes wereobserved when the prolonged-infusion methodof Wheeler and his associates (1, 2) was utilized.Thus, S increased markedly, whereas BSP Tm,decreased during the last half of pregnancy. It isapparent that the Wheeler method provides anextremely useful tool for examining hepatic BSP-removal mechanisms. It not only permits anappraisal of both hepatic uptake and biliary ex-cretion of BSP in quantitative terms, but ob-viously it is capable of detecting significantchanges even when the sensitive clinical BSPtest yields normal results.

The most impressive finding in the presentstudies was the marked rise in S during the lasthalf of pregnancy. There are several possibleexplanations for this change. First, it was neces-sary to demonstrate that significant BSP removalfrom blood in sites other than the liver did notaccount spuriously for the higher values of Sobserved in the last half of pregnancy. Since Swas as high at 24 weeks of gestation as at 40, itseemed unlikely that BSP uptake by the placentaor the fetus, which would enlarge considerablyduring this period of gestation, contributed inany significant way to BSP removal from plasma.This was corroborated by our studies which dem-onstrated that virtually no BSP is taken up by orcrosses the placenta. Others have examinedtransplacental movement of BSP after adminis-tration of a single iv injection of 5 mg per kg.Freiheit detected no dye in cord blood of twoinfants delivered within half an hour of BSPadministration (16). Smith, Moya, and Shnider(17) observed BSP in umbilical vein blood asearly as 20 seconds after BSP injection, and lowlevels of BSP were found in nine of 15 subjects.No BSP was detected in umbilical vein blood ofsix infants. In our constant infusion studies inwhich cord blood was sampled, no measurableamount of BSP was found in 10 of 14 subjects.If a large quantity of BSP had passed into thefetal circulation, it seems likely that relativelyhigh levels of BSP would have been detected,

1438

HEPATIC BSP REMOVALMECHANISMSIN PREGNANCY

since BSP removal from blood is delayed inthe newborn 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 sitesaccounted for a marked increase in BSP removalfrom blood, although this possibility is not ex-cluded. Significant uptake of BSP by tissuesother than the liver apparently does not explainthe rise in S.

Before attributing increased S to an intrinsicchange in the liver, it is necessary to considerother extrahepatic factors that might result inincreased hepatic BSP storage in pregnancy.With the method used in these studies, it is as-sumed that the quantity of BSP taken up intostorage for each increment in plasma concentra-tion of 1 mg per 100 ml is the same over alimited range of plasma concentration (1). It isconceivable 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 with high affinity would take up BSPfirst, thus at low plasma levels. Units withprogressively lower affinity would remain as thequantity of BSP stored increases during risingplasma concentration. Thus lower values for Swould be expected at higher plasma levels. Inthe present studies, average plasma levels ofBSP during each hour of BSP infusion weresomewhat lower in women in the last half ofpregnancy than in control nonpregnant women(5.53, 4.18, and 7.27 mg per 100 ml in pregnantwomen compared to 8.95, 7.01, and 8.88 mg per100 ml in control nonpregnant women, Tables Iand II). Apparently, in the dog, S is relativelyconstant over a range of BSP levels in plasma ofat least 3 to 12 mg per 100 ml (26). Theplasma concentrations attained in the presentstudies fell well within this range, and if S be-haves in the human as it does in the dog, it seemsunlikely that the small differences in plasma levelscould account for the large changes in S observedduring pregnancy.

The degree of protein-binding of BSP in plasmamay exert aIn influence on uptake of BSP intostorage. 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 tototal plasma concentration of BSP. Thus, in-creased unbound BSP in plasma of pregnantwomen 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 revealedno free BSP in plasma 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 plasmaBSP concentrations attained in the present stud-ies did not influence values of S.

A rise in S might be considered to result froma decrease in Tm. This seems unlikely, how-ever, for in at least three situations-in patientswith the Dubin-Johnson syndrome (2), afteracute ligation of the common bile duct in dogs(26), and during administration of 17a-ethyl-19-nortestosterone (37)-S has remained normalwhen Tm, is decreased markedly.

It seems reasonable to conclude, therefore, thatan increase in S during the last half of pregnancyindicates that the relative storage capacity ofthe liver for BSP increases, and furthermore, thatthis alteration is due to an intrinsic change in theliver. Such an increase in S could be accountedfor by an increase in hepatic mass, by increasedstorage capacity of each unit of liver tissue, or byboth. As for increased hepatic mass in normalpregnancy in the human, few data are availablefor analysis. A review of autopsy informationderived from women dying after a brief illnessduring pregnancy in our hospital revealed anaverage liver weight of approximately 1,700 g,with no significant difference in liver mass dur-ing early and late pregnancy. Thus, an increasein size of the liver does not explain the rise in S.Normally, BSP is concentrated in human liverthree to four times above plasma concentration,on the average (2). Although the nature of theconcentrating mechanism is not known-whetherit is a liver protein with greater affinity for BSPthan plasma protein, an active transport mecha-nism, or something else-, an increase in capacityof this mechanism in each unit of liver tissue isthe most likely explanation for the rise in Sduring pregnancy.

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B. COMBES, H. SHIBATA, R. ADAMS, B. D. MITCHELL, AND V. TRAMMELL

The implication of this observation appearsto extend beyond hepatic uptake of BSP. Ob-viously, the human liver is not usually exposedto BSP. A rise in storage capacity for this dyecould hardly be considered a specific response,therefore. It seems reasonable to expect that in-creased hepatic storage capacity for a variety ofcompounds, including drugs, will be found duringpregnancy.

The fall in Tm in the last half of pregnancy mayalso be accounted for by several possibilities.First, if the liver holds on to BSP more avidly,less BSP may be available for excretion. Theobservation that S remained increased at a timewhen Tm was normal in several postpartum pa-tients suggests another mechanism, however.Second, interference with BSP conjugation mightresult in decreased Tm. Previous work indicatedthat conjugation of BSP with glutathione (28-30), a process catalyzed by a liver enzyme (31),does not influence the extent to which BSP isstored in the liver (32). BSP Tm is dependenton conjugation, however, since conjugated BSPis delivered into bile at a faster rate than freeBSP (32). The components of the BSP-gluta-thione conjugating system have not been ex-amined in human liver during pregnancy. Inthe rat, a decrease in BSP conjugating enzymeactivity with normal levels of glutathione hasbeen found during the last 5 days of gestation(14). When liver glutathione levels have beenmaintained, however, a moderate decrease in en-zyme activity has not resulted in a decrease inBSP Tm in the rat (32). Whether conjugationis impaired as a result of a critical decrease inenzyme activity or glutathione content in thehuman liver must await further studies. Ananalysis of BSP compounds appearing in plasmawas carried out in order to detect any majorchanges in conjugation that might occur in thepresent studies. BSP conjugates were observedin plasma during the prolonged infusions of BSP(Tables I to III). The proportion of total plasmaBSP accounted for by BSP conjugates duringeach hour of infusion was almost the same inpatients during the last half of pregnancy and incontrol nonpregnant women (5.8, 21.6, and 20.9%in pregnancy, as compared to 6.4, 26.4, and 24.4%oin controls). The small differences between preg-

nant and control patients in the averages foreach hour were not significant statistically. Sincethe quantity of BSP infused was approximatelythe same in pregnant and nonpregnant subjects,the data above suggest that major changes in BSPconjugation in pregnancy did not affect Tm.

Finally, decreased Tm may result from an im-pairment of the transport processes involved inthe delivery of both conjugated and free BSPfrom liver cells to bile. In this regard, it is ofinterest that a syndrome of obstructive jaundicehas been observed during the latter part ofpregnancy (33-36). After delivery, the obstruc-tive features disappear, and hepatic function re-turns to normal. Obstructive jaundice may recurwith subsequent pregnancies. The nature of theintrahepatic disturbance appears to involve im-pairment of biliary secretory mechanisms. It istempting to speculate that the decrease in BSPTm observed in the present studies reflects asimilar response of the liver, although obviously aless marked one, to changes occurring normallyduring pregnancy. According to this view, ob-structive jaundice might result either from anincrease in quantity of the factor that accounts fordecreased BSP Tm in normal pregnancy, or froma greater sensitivity of biliary secretory mecha-nisms to normal amounts of this factor foundin pregnancy.

Analysis of various mechanisms that mightaccount for both the rise in S and the fall inTm in pregnancy appears to indicate that altera-tions in two independent hepatic systems areinvolved. Furthermore, since the changes inhepatic BSP-removal mechanisms occur duringthe last half of pregnancy, and then return toor toward normal in the first week after delivery,it seems likely that they are mediated by in-creased hormone levels in pregnancy, probablyof 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 quanti-tatively during pregnancy and the first week post-partum in terms of 1) the relative storage ca-

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HEPATIC BSP REMOVALMECHANISMSIN PREGNANCY

pacity for BSP, S, defined as the number ofmilligrams of dye taken up into storage for eachincrement of plasma concentration of 1 mg per100 ml and 2) the maximal excretory rate ofBSP into bile, BSP Tm, in milligrams per minute.S rose 122% during the last half of pregnancy,then returned to or toward normal during thefirst week postpartum. In contrast, BSP Tmdecreased 27% in the last half of pregnancy, thenrapidly increased to normal levels after delivery.These changes appear to reflect alterations intwo independent hepatic mechanisms.

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