Bromsulphthalein and indocyanine green elimination from plasma, and urinary bromsulphthalein excretion, in normal cats

Research in Veterinary Science 1989, 47, 97-101

Bromsulphthalein and indocyanine green elimination from plasma, and urinary bromsulphthalein excretion, in normal cats D. J. MIDDLETON*, Department of Veterinary Pathology, A. D. J. WATSON, Department of Veterinary Clinical Studies, The University of Sydney, New South Wales 2006, Australia The elimination of bromsulphthalein (BSP) and indocyanine green (ICG) from plasma and urinary excretion of BSP were investigated in healthy cats. At 5 mg kg - I, BSP elimination fitted a two-compartment open model, with mean ty,[J of 7·1 (SD 2'5) minutes. A tendency for slower elimination of BSP at 10 mg kg-I suggested saturation of excretory mechanisms, while at 2 mg kg-I accurate dosing and assay of low BSP concentrations were difficult. Urinary recovery of BSP at 5 mg kg-I was O'01 to 0'13 per cent of the total dose. Plasma ICG (0'5 mg kg ' ') data fitted a onecompartment open model, with mean t y, 2'7(sl) 1'0) minutes. In cats, retention tests are more attractive than clearance tests because fewer blood collections are necessary. Proposed reference values are under 3·6 per cent retention of BSP (5 mg kg-I) at 30 minutes and under 17·5 per cent retention of ICG (0' 5 mg kg -I) at 15 minutes. At present economic and technical factors favour BSP over ICG. CHOLEPHILIC dye removal from blood as a measure of liver function can be assessed by either clearance or retention tests. Clearance tests may be more accurate than retention tests as effects of dosage errors and of individual variation in plasma volume are avoided, but a disadvantage is that three or more blood samples are necessary to calculate the overall elimination rate constant (fJ). Bromsulphthalein (BSP) retention tests have been advocated in standard texts for use in the cat but information describing behaviour of the dye in this species is limited (Cantarow and Stewart 1935, Cantarow et al 1938, Smith et al 1940, Center et al 1983). Since BSP was first studied, it has been recognised that, although extensive binding to plasma proteins leads to almost exclusive removal of dye by the liver, small amounts are excreted in urine. The importance of urinary excretion of BSP has generally been considered from the standpoint that loss of large amounts of dye by this route in patients with hepatopathy

·Present address: Department of Veterinary Pathology, Royal Veterinary College, Hawkshead Lane, North Myrnms, Hertfordshire AL97TA

might reduce plasma per cent retention values. Augmented urinary excretion of BSP has been demonstrated in human patients with abnormally high BSP retention in plasma (Norcross et al 1951, Winkler 1961). Thus in cats, urine collection for dye analysis might provide an alternative to retention or clearance tests which necessitate venepuncture. Cantarow and Stewart (1935) gave normal cats 2 mg kg - t BSP and measured up to I mg of BSP in urine after two hours, which is an unusually high proportion of the dose compared with people (under O'5 per cent, Rosenthal and White 1925). There are no data for renal BSP excretion in cats given higher dose rates. For indocyanine green (ICG) clearance tests are more popular than retention tests and the usual protocol in people is administration of O' 5 mg kg- I ICG with calculation of the excretory half-life (t y,[J) or [J (Martin et al 1975). Many authors suggest that lCG is superior to BSP in evaluation of liver function because its plasma decay is less affected by extrahepatic removal (protein-binding of BSP is incomplete), enterohepatic circtilation and elimination into hepatic lymph, and might therefore be a more specific measure of liver function. However, these factors are not of quantitative significance in single injection studies (Wheeler et al 1960). More importantly, BSP removal is impaired in some cases for inapparent reasons (Hunton et al 1960), lCG is not irritant to tissues and side effects of lCG administration are rare (Carski et al 1978). In cats investigation of the disposition of lCG is confined to infusion studies by Larsen (1971) and Kramp and Larsen (1972, 1976) and single injection studies by Center et al (1983). lCG has not been recovered from urine of dogs (Wheeler et al 1958), people (Cherrick et al 1960) or cats (Kramp and Larsen 1976). In this study, the elimination of BSP and lCG was investigated in healthy cats. Three levels of dose of BSP were given, each of which has been recommended in other species. One BSP dose level was also administered to cats to establish baseline data for urinary BSP excretion. Multiple doses were not examined for lCG as only one dose rate (0' 5 mg kg- I) has been suggested generally for clinical use. 97

98

D. J. Middleton, A. D. J. Watson

Materials and methods

Clinically normal adult domestic short hair cats of both sexes, weighing 2'3 to 4·0 kg, were housed in individual cages and given water and nutritionally complete dry cat food (Go-Cat; Carnation) ad libitum. Animals were maintained for at least one month before use. For plasma dye elimination studies, jugular catheters were implanted under general anaesthesia (induced with thiopentone and atropine, maintained on halothane, nitrous oxide and oxygen). Amoxycillin (25 mg) was given intraoperatively. The free end of the catheter was exteriorised on the dorsal neck and covered with a light dressing. Catheters were flushed daily with saline and filled with heparin (1000 iu ml- J) . At least five days after catheterisation and three days before the first dye administration, blood was collected from the catheter for determination by standard methods of packed cell volume, total plasma protein concentration, differential leucocyte count plasma icterus index and for serum protein electro: phoresis. Enzymes assayed were plasma alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase and serum y-glutamyl transpeptidase. These examinations were repeated within nine days of administration of the final BSP dose or the dose of ICG. All doses of dye were delivered from I ml syringes through a 25 gauge needle over 20 seconds into a cephalic vein. Blood (2 ml) for dye determination in plasma elimination studies was collected through the catheter at T = I, 2, 4, 8, 16, 32 and 64 minutes after the beginning of injection (T = 0) and anticoagulated with lithium heparin (50 iu). Plasma was assayed for BSP concentration within six hours of collection using the method of Seligson et al (1957)at 580 nm on a Perkin~lmer ~ 24 doub~e beam spectrophotometer. A change in optical density occurred after addition of acid reagent to the zero time specimen (which did not contain BSP) and this was subtracted from changes in optical density of subsequent samples in calculating Expressing the amount of dye dye ~o?ce~tration: rernammg In the circulation as percentage retention rather than dye concentration is conventional, and assumes initial dispersion of dye into a plasma volume of 50 ml kg Fresh aqueous BSP standards of 20 50 and 100 mg litre-I were used as appropriate,' to represent 50 per cent retention of 2,5 and 10 mg kg-I BSP, respectively. An ICG standard (5 mg litre-I) representing 50 per cent retention was made using pooled dog plasma. Each eat's control plasma was assayed as the plasma blank. Plasma concentrations of ICG were read directly at 805 nm in a Unicam SP500 Series 2 spectrophotometer with infrared photocell. Plasma r

specimens (0' 5 ml) were diluted with I ml O·9 per cent sodium chloride before assay. Statistical analyses of the data are described with the results.

Experiment 1 Six cats were deprived of food for six to 12 hours baseline blood samples were collected from the catheter and BSP (Hynson, Westcott and Dunning) was given in a 50 g litre-I solution. The dose of BSP was 2, 5 or 10 mg kg ' ' given at weekly intervals according to a duplicated 3 x 3 orthogonal Latin square (each cat receiving each dose level once).

Experiment 2 Haematological and biochemical examinations were performed in the week before BSP dosage. Seven cats were deprived of food for six to 12 hours, then received 5 mg kg- I BSP as a 50 g litre - I solution. After injection, each cat was placed in a catbox to discourage urination. Two hours after BSP administration general anaesthesia was induced and maintained with halothane nitrous oxide and oxygen through T-piece and fac; mask. The urinary bladder was catheterised and emptied, and urine volume measured. Routine urinalysis was performed on a sample and the remainder was centrifuged and BSP assayed in the supernatant. An aqueous BSP standard of 5 mg litre-I was freshly prepared. A measurement for urine BSP blank was obtained from the mean difference in optical density of urine from 10clinically normal young adult cats of both sexes before and after the addition of acid reagent to urine diluted I: I with alkaline buffer (Seligson et al 1957). Urine of pH ~ was found to be adequately buffered by the alkaline reagent.

Experiment 3 Six cats were deprived of food for six to 12 hours baseline blood samples were collected from the catheter, and lCG (Cardiogreen; Hynson, Westcott and Dunning) was given at O: 5 mg kg- I as a freshly prepared 5 g litre " ' aqueous solution.

'.

Results Experiment 1 Pre- and post-treatment haematology and biochemistry were unremarkable. All cats remained clinically healthy. Intravenous administration of BSP to cats was followed immediately on two occasions by vomiting (one cat at both 5 and 10mg kg- I) and twice by trembling with hyperaesthesia (at a dose level of 2

Bromsulphthalein and indocyanine green in cats

five per cent confidence limits for predicted BSP at 30 minutes after administering 5 mg kg -I of dye were O' I to I· 6 mg litre - I, or up to I . 6 per cent retention when using a control sample of plasma. In the absence of a control specimen, the range became up to 3· 6 per cent retention, after compensating for the difference in optical density produced by the addition of acid reagent to buffered normal cat plasma (plasma blank).

1000

o 10mg kg- 1 Ii 5 mg kg- 1 02 mg kg- 1

100

~

99

10

Cl

E

0. en 1 rIO

Experiment 2

0·1

0'01 12 4

8

32

16

Time (min)

FIG 1: Plasma BSP concentrations in six cats at various times after being given 2, 5 and 10 mg kg - , BSP at T = O. Data points are the geometric mean of six values with 95 per cent confidence limits as the measure of spread. The Y axis is a logarithmic scale

mg kg" ' in the cat noted above as vomiting and in another cat receiving 5 mg kg-I). Perivascular leakage of BSP was not apparent. BSP was not detected at 64 minutes in five cats at dose levels of 2 and 5 mg kg- J so plasma concentrations at this time were eliminated from statistical analysis. Six of the remaining 108specimens were not collected. Fig I shows semi-log plots of plasma BSP against time for each dose rate. Correlation coefficients for linear regression equations calculated for each dose using log transformed BSP concentrations on time for one, two and four minutes (distribution phase) and for eight, 16 and 32 minutes (elimination phase) were statistically significant at the 1 per cent level. The (3 or elimination phase (eight to 32 minutes) of the plasma disappearance' curve was examined further and the homogeneity of the three (dose levels of 10, 5, 2 mg kg-I) regression coefficients was assessed (Student's t test). For each pair P>0'05, however, there was a tendency (0' 2>P>O· 1) for the 10 mg kg- I dose to be eliminated more slowly. This is consistent with saturation of the BSP excretion pathway but the significance of the result should be tested further in a larger number of cats. At 2 mg kg- 1 accurate dosage .was. hard to achieve and the lower BSP concentrations produced were difficult to assay. Thus, from the 5 mg kg-I dose level data, the slope (b) of the regression equation for each cat was calculated (Steel and Torrie 1960) to determine the excretory half-life (t'l,(3) of the drug, where t'l,(3

=

0'693 -(3- and (3

=

b -1-oglQe

(Baggot 1977). The mean t'l,(3 was 7· 1 (so 2· 5) minutes. Ninety-

Haematological and biochemical findings were unremarkable. No cat reacted clinically to BSP administration or urinated in the two hours after BSP administration. Urine specific gravity and BSP determinations are displayed in Table 1, with estimates of the percentage of the given dose excreted in the urine within two hours. Urinary excretion of BSP was low: up to l : 5 mg Htre: I or 0·13 per cent of the dose. There was no useful correlation between urine BSP concentrations, the percentage of the dose excreted in urine and urine concentration. Experiment 3 Haematological and biochemical findings in cats receiving lCG were unremarkable. No untoward reactions were observed after administration of lCG and no known perivascular injections were made. Two of 42 specimens were not collected. ICG was detected in five cats at 64 minutes, but at concentrations equal to or greater than ICG at 32 minutes, and at the limit of sensitivity of the assay. Accordingly, linear regression analysis was restricted to log transformed ICG between one and 16 minutes. The ICG data fitted a one-compartment model (Fig 2). For each cat t'l, was calculated as in experiment 1. The mean t y, was 2·7 (so 1'0) minutes. The 95 per cent confidence limits on the predicted ICG at 15 minutes were from 0 to 1· 75 mg litre : ", or up to 17·5 per cent retention in normal cats. . TABLE 1: Urine concentration. and amounts of bromsulphthalein (ssp) dye excreted in urine within two hours of intravenous administration at 5 mg kg - 1 to seven normal cats

Urine specific gravity

Concentration of BSP in urine Img litre-')

1·040 1·033 1·052 1·040 1·041 1·033 1·051

0·7 0·3 0·7 0·1 0·2 1·4 1·5

Absolute amount of BSP Percentage of in urine administered Img) dose in urine 0·014 0·005 0·013 0·002 0·004 0·020 0·008

0·09 0·03 0'11 0·01 0·03 0·13 0·04

D. J. Middleton, A. D. J. Watson

100 10

1 0' 04

0·1

1 2

4

8

rime (mini

16

FIG 2: Plasma ICG concentrations in six cats at various times after being given 0'5 mg kg- 1 ICGat T ~ O. Data points are the geometric mean of six values with 95 per cent confidence limits as the measure of spread. The Y axis is a logarithmic scale

Discussion

Side effects observed in two cats after BSP administration probably represent individual sensitivity to the drug (one cat reacted at three dose levels) and resemble the nausea described by Magath (1935) in heavy people given 5 mg kg- 1 BSP rather than the anaphylactic or anaphylactoid reactions reported after extravasation or previous administration of BSP (Shafer and Gregory 1968), or the reaction noted in asthmatics (Stecher 1959, Venger 1961). Both cats received BSP on later occasions without complication. BSP data fitted a two-compartment open model in which the first exponential delineated the a or distribution phase (during which elimination may also take place) and the ~ or elimination phase portrayed the irreversible movement of drug from the central compartment. Two-compartment models have been fitted satisfactorily to BSP plasma decay curves in dogs (Richards et al 1959) and people (Barber-Riley et al 1961, Forker and Luxon 1978)although the shapes of the curves differed slightly between species. In cats, the 10 mg kg-I dose level of BSP tended to be excreted more slowly than lower doses. Similarly, Winkler and Gram (1961) found BSP elimination in people slowed as the dose administered increased from 5 to 10 to 15 mg kg- I. Dose-dependent elimination results from saturation of biotransformation reactions, carrier-mediated transport (including biliary secretion) or protein-binding (Baggot 1977). Accordingly, a dose level of IOmg kg-I BSP might not be sufficiently sensitive for diagnostic use in cats. Cantarow and Stewart (1935) found BSP retentions of o per cent at 30 minutes in normal cats given single doses of 2 mg kg -I, and the present authors found no plasma BSP at 32 minutes in four of six cats given doses of 2 mg kg- I. It would seem essential to have at

least a low concentration of BSP present in normal cats at the sampling time for comparative purposes. The 5 mg kg' ' dose was more suitable in this regard and did not exceed the BSP excretion capacity of the normal liver. Examination of data provided by Center et al (1983) suggested a tY2~ of approximately 13minutes in cats given BSP at 5 mg kg- I. In comparing the t 'Ii~ of 7' 1 minutes provided by the authors' data, it should be noted that BSP at 32 minutes in this study was lower than the BSP at 30 minutes of Center et al (1983). Center et al (1983)did not describe the use of a plasma blank for BSP in each cat and it is possible that, at low BSP, their recorded changes in optical density consisted largely of a plasma component, leading to spuriously high BSP estimations. If 'static' terms such as percentage retention at 30 minutes (t30) are considered, this problem may be avoided as omission of control specimens is taken into account. The authors' suggested cat reference of t 30 up to 3· 6 per cent BSP retention agrees well with the under 3 per cent found by Center et al (1983). The amount of BSP excreted in the urine of normal cats in two hours after a dose of 5 mg kg:", that is, up to 0·13 per cent of the dose, is less than that found in similar tests in people (lngelfinger et a11948, Richards et al 1959). Absence of correlation between urine BSP concentration and the fraction of the BSP dose excreted by the kidney in cats means that the bladder must be emptied completely for calculation of total excreted BSP. At least seven plasma half-lives (50 minutes) should elapse after BSP dosage before urine sampling is done to allow virtually complete renal dye elimination. However, a longer urine collection period may be clinically preferable as t 'Ii~ can be prolonged by hepatopathy thus protracting the appearance of BSP in urine (Winkler 1961). Disposition of ICG in plasma "after intravenous injection in cats fitted a one-compartment open model. Center et al (1983) found two compartments more appropriate for cats receiving 1· 5 mg kg The mechanism of excretion .of ICG is such that a onecompartment model might be expected as there is no reflux of ICG from liver cell to plasma (Vogin et al 1966), no intrahepatic resorption from bile into lymph (Hunton et a11960) and minimal enterohepatic circulation (Wheeler et aI1958), all of which if present would contribute to a distinct a phase. The time at which the single blood sample should be taken in lCG retention tests is not well established as clearance tests are often done. Center et al (1983) suggested t 30 for cats given 1· 5 mg kg-I of 7· 3 (SD 2'9) per cent. In many cats given 0'5 mg kg-I, blood collection later than 15 minutes will produce difficulty in assay of lCG, so the time of 15minutes with predicted retention of under 17' 5 per cent (or ICG up to I· 75 mg Iitre : ') in 95 per cent of normal cats is proposed. r

'.

Bromsulphthalein and indocyanine green in cats However, putative advantages of ICG over BSP are not clearly outweighed by the requirement for a spectrophotometer with an infrared photocell, standards which contain albumin, and additional costs. At the time of this study, ICG at 0'5 mg kg- I was six times as expensive per cat as BSP at 5 mg kg r

'.

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MAGATH, T. B. (1935) American Journal ofDigestive Diseasesand Nutrition ll, 1-4 MARTIN, J. F., MIKULECKY, M., BLASCHKE, T. F., WAGGONER, J. G., VERGALLA, J. & BERK, P. D. (1975)

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Received December 9, 1987 Accepted September 29, 1988