Bromsulphalein (BSP) clearance in ageing rats

Exp. Geront. Vol. 3, pp. 147-153. Pergamon Press 1968. Printed in Great Britain

BROMSULPHALEIN

(BSP) C L E A R A N C E

IN AGEING

RATS*

C. F. HOLLANDER,F. R. DE LEEUW-ISRAELand J. M. ARP-NEEFJES Experimental Gerontological Unit of the Organization for Health Research, TNO, Lange Kleiweg 151, Rijswijk, Z. H., The Netherlands INTRODUCTION OBSERVATIONSby several authors suggest that the ageing process affects the morphological characteristics of the liver cells of various species (Bucher, 1963 ; Carr, Smith and Keil, 1960). Changes in the liver of the rat with age consist of a gradual shift from a diploid parenchymal cell population at birth to a polyploid cell population with over 80 per cent polyploid cells (Bucher, 1963 ; Post, Klein and Hoffman, 1960). Previous investigations have indicated that polyploidy of liver cells is an ageing process (Hollander and Thung, 1966). The objective of the present study was to determine the effect of ageing on liver function and to investigate if a relation exists between functional and morphological changes during ageing. The bromsulphalein method was employed as a parameter of liver function (Mateer, Baltz, Marion and MacMillan, 1943). Sulfobromophthalein-sodium, hereafter referred to as BSP, is an indicator dye, which is colourless at a pH below 7.4 and purple at a pH above 10.4. A diagram of the metabolism of BSP is presented in Fig. 1. After intravenous administration, BSP is bound by plasma albumin and by plasma ~t-lipoproteins Blood

Liver Cell

Unconjugated BSP + albumin or al lipoproteins

Bile

Unconjugated BSP ~BSP q- glutathione

'~ Conjugated BSP

j-

+ Enzyme

Fit. 1. Diagram of BSP metabolism. (Baker and Bradley, 1966; Baker, 1966). BSP is selectively removed from the bloodstream by the liver of many vertebrates. Probably, hepatocellular uptake of the dye occurs directly from the BSP albumin complex that has moved across the sinusoidal wall (Baker and Bradley, 1966). Whether the liver removes the portion of the dye associated with the ~t-lipoproteins in a different manner of at a different rate from the albumin bound moiety has not been ascertained (Baker, 1966). After entering the

F

* These experiments were done in the Netherlands Cancer Institute, Amsterdam. 147

148

C. F. HOLLANDER, F. R. DE LEEUW-ISRAEL AND J. M. ARP-NEEFJES

parenchymal cells of the liver a considerable amount of the dye is conjugated with glutathione, a process catalyzed by the BSP glutathione conjugating enzyme (Combes and Stakelum, 1961 ; Combes and Stakelum, 1962). Subsequently free and conjugated BSP are excreted into the bile. In the rat BSP conjugates account for most of the BSP appearing in bile (Krebs and Brauer, 1958). The removal of BSP from the plasma involves several processes: uptake, conjugation and storage of the dye in the liver followed by biliary excretion. Saturation of the hepatic storage compartment can occur both after a single intravenous dose and after continuous intravenous administration of the dye (Bonfils, Bernad~s and Hall&Pannenko, 1966; Wheeler, Meltzer and Bradley, 1960). After saturation, the rate of uptake from the blood is limited by the biliary excretion mechanism. Because BSP is excreted with the bile and the dye does not re-enter the general circulation, BSP clearance is generally employed in the physiological and clinical study of liver function. MATERIALS AND METHODS Female albino rats (strain R-Amsterdam), maintained on commercial food pellets (Kraay A.M. pellets) and tap water ad libitura, were used in the experiments. Three groups of rats have been employed for these studies. The first group consisted of 3-, 6and 9-month-old rats. These animals were used to establish the technique. The second group consisted of eleven 30-month-old rats (mean body wt. 235 g). These rats were tested only once and afterwards killed and autopsied. In the third group a longitudinal investigation on 8 rats was performed: the tests were done at the ages of resp. 3 months (mean body wt. 180 g), 6 months (mean body wt. 200 g), 9 months (mean body wt. 210 g) and 12 months (mean body wt. 230 g). The bromsulphalein was obtained from Merck-Darmstadt (Bromthalein ~ 0.5 g in 10 ml). A single dose of 5 mg BSP per 100 g body wt. was injected into the tail vein, the rat being under light ether anesthesia (Van der Hem, 1964; Van der Vies, 1966). Blood samples were taken at different time intervals after injection of the dye. The blood sample was obtained by cutting the tail with a razor blade. The tests were done on the Ultramicro Analytical System (Beckman), providing reproducible measurements of drop-size volumes, viz. only 20/~I serum is needed. BSP in the serum was measured colorimetrically after dilution and alkalization of 20/~1 of the serum with 200/~l of NaOH 0.5 N in NaC1 0.9 per cent against a blank of a sample obtained prior to the injection and treated exactly as the specimen samples that were assayed. Turbid or hemolyzed sera were discarded. The readings were made in a Beckman model 153 spectro-colorimeter of the Ultramicro Analytical System at 580 m/z. The colour intensity of different batches of BSP can vary. Therefore it is necessary to make up a standard for each batch. The standards were prepared by diluting the BSP solution from the ampoule used for the intravenous injection, with NaC1 0.9 per cent. The results were expressed in rag/100 ml of serum. A brief discussion of the BSP method seems appropriate. In defining base line values of normal liver function, the weight of the liver has to be taken into account. The literature does not provide conclusive data covering the age and the weights of the body and the liver. Therefore we had to collect our own data. Four 3-, 6- and 9-month-old female rats were killed with ether and the body was weighed. The abdominal cavity was opened and the liver was removed. It was allowed to drain on filter paper for several

BROMSULPHALEIN (BSP) CLEARANCE IN AGEING RATS

149

minutes, any adhering clots were removed and the liver was weighed. In this manner liver: body weight ratios were found, which varied between 3.1-3.2 per cent. These data were comparable with the results of Barrows (1965), who did not find a significant difference in the liver: body weight ratios of young and old male rats.

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a~

~

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1"7 "~I 2.4 -I- 0 " 9

0 !

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I0

15

MINUTES

20 AFTER

25

30

INJECTION

FIG. 2. Bromsulphalein (BSP) saturation curve of 3 month-old female R rats. The mean value of BSP retention and standard deviation of 4 rats is presented. The retention in mg % is plotted on a semilogarithmic scale. Time is given in minutes after injection of BSP. The control BSP clearance studies were performed on four 3-month-old rats, which received a single intravenous injection of 5 mg B S P / 1 0 0 g body wt. The change in serum concentration with time is shown in Fig. 2. The form of this curve is similar to that seen in other animals and man in that it shows a saturation point (the point of deflection) and is called a saturation curve (Ingelfinger, Bradley, Mendeloff and Kramer, 1948). Initially the liver contains no dye and the first phase of the graph is predominantly due to the rapid uptake of the dye by the liver from the plasma. This is followed by a gradual steady fall in plasma concentration which is coincident with the continued excretion of the dye into bile (Richards, Tindall and Young, 1959). This experiment showed that the given dose of BSP (5 mg BSP/100 g body wt.) was sufficient to saturate the liver compartment and to leave dye in the blood whose rate of uptake is limited by the biliary excretion mechanism (excretory phase = second phase of the graph). Additional BSP clearance curves were determined in animals of the ages of resp. 3, 6, 9 and 12 months. The plasma levels at zero time were determined by

150

C. F. HOLLANDER, F. R. DE LEEUW-ISRAEL AND J. M. ARP-NEEFJES

extrapolation of the initial parts of the curves plotted on semi-log, paper. These plasma levels showed no significant difference. The differences found between the excretory phase of the curves at different ages will be discussed in the next paragraphs. No signs of toxicity after repeated BSP administration were found. In the other experiments the blood samples were taken solely in the excretory phase, 15, 30 and 45 min after injection of the dye, and the BSP concentrations were plotted against time on semi-logarithm paper. RESULTS BSP "excretion curves" of 3-, 6-, 9- and 12-month-old rats and of 30-month-old rats are given in Fig. 3. The higher BSP retentions found in the 30-month-old rats compared to the retentions in the 3-month-old rats may indicate a physiological impairment of liver function in the old rats. However, the 30-month-old rats were affected by mycoplasma pneumoniae (PPLO), causing chronic respiratory disease (CRD). As a result of this disease liver congestion occurs. At autopsy, chronic bronchitis, bronchiectasis, emphysema and venous stasis of the liver were observed in all 30-month-old rats. Some animals showed ~o 2O © :5 z

~

•

3 MONTHS

•

6

"

(8)

(~)

14

7" 0 ~ IOZ ~-u.l

8-

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4"

2-

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!

1

15

30

45

MINUTES AFTER I N J E C T I O N

FIG. 3. Bromsulphalein (BSP) clearance in female R rats at different ages. T h e retention in m g % is plotted on a semilogarithmic scale. T i m e is given in minutes after injection of BSP. N u m b e r of animals used per age group is given in parenthesis. Standard deviations at 15, 30 and 45 rain are resp. : A2"2, 0"9, 0"9; • 1-9, 1 "2, 0"5; • 2.6, 2-3, 2.1 ; • 2"2, 2"9, 0"7; [] 3"3, 1 "8, 1"1.

BROMSULPHALEIN (BSP) CLEARANCEIN AGEING RATS

151

also signs of bronchopneumonia with small lung abscesses. In human patients with passive liver congestion BSP retention is often slightly increased (Moyer and Wurl, 1951). Nevertheless, it seems improbable that liver congestion did influence the BSP retention in the old rats because the 6-, 9- and 12-month-old rats, clinically not affected by PPLO, showed the same values. Preliminary results indicate that the change in BSP excretion with increasing age could differ in various inbred strains of rats. DISCUSSION This study indicates that there exists a change in BSP excretion of the liver with age as indicated by the higher BSP retentions found in the animals of 6 months of age and older as compared to 3-month-old animals. This change occurs between the ages of 3 and 6 months and can hardly be called an ageing phenomenon. The "excretion curves" (Fig. 3) of the 6-, 9-, 12- and 30-month-old rats run practically parallel to each other and to the "excretion curve" of the 3-month-old rats, indicating the same rate of excretion of dye into bile. Therefore, it is not likely that ageing does alter the capacity of the liver cell to excrete BSP into bile. This is in accordance with the findings of Thompson and Williams (1965) in man. The question arose of the change in BSP excretion could be explained by a change in storage capacity of the liver cells. In 3-month-old animals the minimum dose of BSP needed to get a saturation curve is 5 mg/100 g body wt. (standard dose used in these experiments). In female R rats of different ages an investigation was done to establish the minimum dose of BSP needed to get a saturation curve in each age group (age dependant dose). The outcome of this study is given in Table 1, in which the given values are those of the "excretory phase" of the saturation curves. There exists a gradual decrease in minimum dose needed to obtain a saturation curve with age. After the age dependant dose is given, no difference in BSP retention between the age groups exists. This indicates that the storage capacity of the liver is gradually changing with age. A possible explanation for the changes found, could be the polyploidization of the liver cells. Bucher (1963) stated that the polyploidy shift of the liver cells in rat stabilizes around the age of 6 months. After this age only a slight change in ploidy of the liver cell population occurs. At present no proof exists for the supposition that polyploidization TABLE 1. COMPARISONBETWEENTHE EFFECTS OF A STANDARDDOSE (5 mg/100 g bw) AND AN AGE DEPENDANT DOSE OF BSP IN FEMALER RATS OF DIFFERENTAGES Standard dose Age in months 3 6 9 12 15

Age dependant dose

mg % BSP in plasma after 15 rain 30 rain 45 rnin 4-8~0"9 11-9±2"1 14-9~2.4 18"0±3-6 15-8~2-1

2-3±0"4 4"2±0.8 7"2±1"2 6"8±1"3 5"7±0"7

1.1 ~ 0 " 1 1.7±0'2 3.5±0'1 2.6±0"4 2.9±0"4

m g % BSP in plasma after 15 rain 30 rain 45 rain 4"8±0"9 5.4±0'8 6.6±1"2 4"9±1-3 5.2±0"9

2"3 ± 0 " 4 2"3±0"3 2.1±0-4 2-4±0"5 2'5~0"2

1.1 ~ 0 . 1 1"2±0'1 1-1±0.2 1.2±0.3 1"0±0"1

(mg/100g bw) 5 4 4 3 3

Mean values of BSP retention in plasma at different time intervals and S D of 4 rats in every age group are given.

152

C. F. HOLLANDER, F. R. DE LEEUW-ISRAEL AND J. M. ARP-NEEFJES

can deteriorate or improve the "efficiency" of the liver cells (Epstein, 1967). This suggests that there is no functional impairment of the single liver cell with age, but that the decrease of BSP excretion could be explained by a possible decrease of the n u m b e r of liver cells, thus assuming not a qualitative but only a quantitative change. Suggestive for this hypothesis are the findings of Bucher and Glinos (1950), who found that the average n u m b e r of hepatic nuclei per cubic millimeter of liver tissue are less in older rats as in younger animals. T h i s is due to polyploidization of the liver cells. In this respect, the liver can be compared with other organs or organ systems (Makinodan and Peterson, 1964, 1966 a, b; Wigzell and Stjernsw~ird, 1966; Tonna, 1966) in which was found that functional cells of a senile animal do not differ from that of a young animal. We agree with Makinodan and Peterson (1964) that one fruitful approach to understanding senescence is to put greater emphasis on the decrease in pool size of vital cells, rather than on decrease of function of the cells, as is commonly done.

Acknowledgement--We are grateful to Dr. J. VAN GOOL, Department of General Pathology, University of Amsterdam for his hospitality in allowing us to use the Ultramicro Analytical System (Beckman) in his Department and for his continued interest in our work.

REFERENCES BAre'R, K. J. and Bm~DLmr, S. E. (1966)J. clin. Invest. 45, 281. BAKER, K. J. (1966) Proc. Soc. exp. Biol. Med. 122, 957. B~u~ows, C. H. (1965) In Aging and levels of biological organization. (Edited by BRUES, A. M. and SAcrm~, G. A.), p. 154. University of Chicago press. BONFILS, S., BEm'~AD~, P. and HALLC.-PANN~O,O. (1966) Path. Biol. 14, 903. BUCHER, N. L. R. (1963) Int. Rev. Cytol. 15, 245. BUCHER, N. L. R. and GLINOS, A. D. (1950) Cancer Res. 10, 324. C~u~, R. D., SMITH, M. J. and KEn., P. G. (1960) ~lrchs. Path. 70, 1. COMBES, B. and ST~m~LUM,G. S. (1961)J. din. Invest. 40, 981. COMBES, B. and STAm~LUN,G. S. (1962)ft. din. Invest. 41, 750. EPSTF.IN, C. J. (1967) Proc. natn. Acad. Sci. U.S.A. 57, 327. HEM, G. K. VAN rmn (1964) Thesis, Groningen. HoLi.~rmR, C. F. and Tatn~c, P. J. (1966) In Radiation and Ageing. Proc. of a Colloquium held in Semmering, Austria. June 23-24, 1966. (Edited by LINDOP, P. J. and SAen~, G. A.). pp.3-14. Taylor and Francis, London. INCET.FINCF.a,F. J., Bm~DLE'L,S. E., MENDRT.OFF,A. I. and Km~MER,P. (1948) Gastroenterology 11, 646. KReBS, J. S. and Bm~Up.R,R. W. (1958) Am. J. Physiol. 194, 37. MAKINODAN, T. and P~rmtsoN, W. J. (1964)o7. Immun. 93, 886. MXKINOD~, T. and t~T~SON, W. J. (1966) Devl. Biol. 14, 96. MAKINOD~, T. and Pm'ERSON, W. J. (1966) Devl. Biol. 14, 112. MAX~.Ea, J. G., BALTZ,J. I., MARION, D. F. and MAcMIr.T.AN,J. M. (1943) ft. Am. me& Ass. 121, 723. Mo'/~R, J. H. and WURL, O. A. (1951) Am. ~. reed. Sci. 221, 28. PosT, J., KLEIN, A. and HOFFM~U'~,J. (1960) Archs. Path. 70, 314. RICI~AaDS, T. G., TtNDALL,V. R. and YOUNG,A. (1959) Clin. Sci. 18, 499. TgOMPSON, E. N. and WmUAMS R. (1965) Gut 6, 266. TONNA, E. A. (1966) Periodontics 4, 105. Vt~° J. VAN D~R (1966) Personal communication. WHirleR, H. O., Me~-Tzm%J. I. and B~rmEY, S. E. (1960) ft. din. Invest. 39, 1131. Wmzeu., H. and STJm~NSWgRO, J. (1966) J. natn. Cancer Inst. 37, 513.

BROMSULPHALEIN (BSP) CLEARANCEIN AGEING RATS S u m m a r y - - L i v e r function in ageing rats was studied, using the bromsulphalein (BSP) clearance test. T h e test was done on ultramicro scale. This made it possible to repeat the test several times in the same animal and to start a longitudinal study. In 3-month-old rats the BSP retentions, measured 15, 30 and 45 min after a single injection of the dye, were significantly lower than in 6-, 9-, 12- and 30-monthold rats. T h e values of the 6-, 9-, 12- and 30-month-old animals d i d not differ significantly. T h e results of this study are discussed with regard to the change in liver cell population with increasing age, viz. polyploidy and change in number of parenchymal cells.

R 6 s u m 6 - - O n a 6tudi6 la fonction h6patique de rats vieillissants en se servant de l'6preuve d'6puration de bromosulpha16ine (BSP). L'gpreuve 6tait pratiquge 6chelle ultramicroscopique. Ceci permettait de r6p6ter l'6preuve plusieurs lois chez le m~me animal et de mettre en route une 6rude longitudinale. Chez les rats de 3 mois, le taux de r6tention de BSP, mesur6 15, 30 et 45 minutes aprbs une injection unique du colorant, fut significativement plus bas que chez les animaux de 6, 9, 12 et 30 mois qui ne diff6raient pas significativement entre eux. Les rgsultats de cette 6tude sont discut6s en vue de la modification darts la population de cellules h6patiques avec l'gge avan~ant, notamment de la polyploidie et une modification du nombre de cellules parenchymateuses.

Z u s a m m e n f a s s u n g - - B e i alternden Ratten wurde mit Hilfe des Bromsulphalein (BSP)-Clearancetests die Leberfunktion untersucht. Der Test wurde im Ultramikromal3stab durchgefiihrt. Man konnte deshalb den Test bei demselben T i e r mehrfach wiederholen und eine Verlaufsstudie unternehmen. Bei 3-Monate-alten Rat-ten waren die BSP-Retentionen, die 15, 30 und 45 Minuten nach Injektion des Farbstoffs gemessen wurden, signifikant niedriger als bei 6, 9-, 12- und 30-Monate-alten Ratten, welche sich nicht signifikant unterschieden. Die Ergebnisse dieser Untersuchung werden im Hinblick auf Veriinderungen der Polyploidie und der Zahl der Parenchymzellen in Leberzellpopulationen zunehmenden Alters diskutiert.

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153