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Bromsulphthalein fractional clearance in normal beef cattle
CLINICA
CHIMICA
ACTA
BROMSULPHTHALEIN
I33
FRACTIONAL
CLEARANCE
IN NORMAL
BEEF
CATTLE
S. E. HUNT
AND P. J. McCOSKER
Department of Clinical (Received
June
Igth,
Studies, Veterinavy School, University of Queensland (Australia) * 1967)
SUMMARY
Application of the BSP fractional clearance test to beef cattle, has been studied. A simplified method for the collection of accurately timed serial blood samples free of haemolysis is presented. This involves the use of an indwelling cannula (Braunula). Elimination of haemolysis simplifies spectrophotometric estimation of BSP concentration, as no correction for contamination by haemoglobin is necessary and readings may be made at one wavelength (580 mp). Mean BSP fractional clearance of 25 beef cattle (200-522 0.166 & SD 0.034, with half life (T&) 4.18 min. No significant served. The reproducibility of results using this method analysis of variance of the data shows that the day-to-day is not significant.
lbs body weight) was sex difference was ob-
has been examined and an variation within individuals
The different excretion pattern observed in normal cattle, namely single-phase as compared with biphasic, which occurs in other species, has been discussed.
INTRODUCTION
Rosenthal and White’ developed the original Bromsulphthalein (BSP) liver function test in human medicine. In their test, the percentage of dye remaining in the plasma at 5 and 30 min from the time of intravenous injection of BSP at the rate of 2 mg per kg body weight, was used as an index of liver function. Application of BSP retention as a test of liver function in cattle has not always yielded satisfactory results 10--15.A liver function test based on this method is currently being used as a satisfactory index of liver function in the dog2-7. Drill and Ivy8 compared the BSP retention test with serum alkaline phosphatase, prothrombin time, galactose tolerance and indirect Van den Bergh estimations. They concluded that BSP retention was the most sensitive of these tests. Svirbely et al9 found that the BSP retention test was more sensitive than Rose Bengal retention and more reliable than bilirubin estimation or urinary urobilinogen. * Mailing
addrex:
Mill Road,
St. Lucia,
Queensland. Clin.
Chim.
Acta, 18 (1967)
133-140
HUNT, MCCOSKER
I34
Ingelfinger et al. I6 have shown that, in man, concentration of dye in the plasma after intravenous injection of a single dose, declines exponentially and a straight line is obtained when log, of concentration is plotted against time. This has been confirmed by Lavers et 61.” and Goodmanis, who suggested that the slope of the line so produced would provide a quantitative measure of liver function. This function has been termed “fractional clearance” by Lewisls~zo who defined this term as “the fraction of the total fluid volume (or solvent compartment) cleared in unit time”. Mixner and Robertson2* applied the principle of fractional clearance of BSP to the study of normal liver function in dairy cattle. They found that over the period 3-15 min the results obeyed the exponential law suggested by Ingelfinger et al.16. The mean BSP fractional clearance for nine Holstein bull calves and five lactating Holstein cows was 0.156 and 0.1gz respectively. Cornelius et aLz2 record results of BSP fractional clearance for fifteen non-lactating dairy cows as, 0.22 & 0.03, and for five Iz-r&month-old Hereford steers and heifers as 0.155 & 0.011. Hansenz3 was unable to confirm that BSP is excreted exponentially in cattle. The object of this paper is to present the results obtained for BSP fractional clearance in normal beef cattle. MATERIALS AND METHODS
Animals
Twenty-five normal yearling Hereford or Hereford cross cattle, zoo to 522 lbs. body weight, were examined. They comprised twelve entire males, nine entire females and four steers. They were housed in metabolism cages and a settling in period of one week was allowed before experiments were commenced. Throughout the experimental period, water and lucerne chaff were supplied ad lib. In addition, six similar animals, 300-350 lbs. weight, were examined three times on consecutive days under identical conditions to ascertain the repeatability of the method. All six animals were males, four being Shorthorn-Hereford crosses and two Herefords. Fractional
clearance
technique
The determination of fractional clearance is dependent upon collection of accurately timed blood samples, free of haemolysis or turbidity. The method of collection outlined below, ensured that accurately timed blood samples free of haemolysis were obtained. Further, the variable degree of turbidity which occurred in heparinised plasma samples was eliminated when serum was collected instead. Infusion
and collectiort
of samples
A 5% aqueous solution of BSP* was used. Distilled water was used in preference to physiological saline as it was found that BSP precipitated out of solution in the latter at room temperature. This solution was sterilised by autoclaving. Infusion and collection of samples was facilitated by the introduction of a “Braunula zL”** into the jugular vein. A small area over the jugular vein was shaved * Sulphobromophthalein Sodium salt. Koch-Light Laboratories Ltd. Code No. 51~4h. ** Manufactured by K. Braun, Melsungen, W. Germany. Obtainable in Australia from Boots Pure Drug Co. Ltd. (The “Braunula” comprises three components: an outer plastic cannula, a 34” needle insert which protudes $” beyond the plastic cannula and a plastic plug, the end of which is sealed with a gum rubber cap.) CZin. Chiwz. Acta,
18 (1967) 133-140
BSP
CLEARANCE
and cleaned
I35
IN CATTLE
with 0.1%
Cetavlon.
Local
anaesthetic
(3% Citanest)
was infused
into
the area and a stab incision was made with a scalpel. The “Braunula” was inserted into the jugular vein via the stab incision, and its needle insert withdrawn, leaving the plastic cannula in place in the vein. An initial blood sample was collected by allowing blood to drain into a r5-ml centrifuge tube. The “Braunula” was then sealed with the plastic plug and a few ml of heparinised physiological saline infused through the gum rubber cap to prevent clotting. Infusion of the BSP (I mg/lb. body wt.) was made through the cap. A stopwatch was started at the midpoint of injection. A few ml of heparinised saline were infused in a similar manner to ensure that all the BSP had entered the vein and also to prevent blood clotting in the cannula. This was repeated immediately after each sample collection. Blood samples were collected every 4 min for periods up to 24 min, by removing the plastic plug and allowing blood to flow directly into r5-ml centrifuge tubes, after ensuring that all the heparinised saline in the cannula had been removed.The time was noted at the midpoint of collection. Centr
The estimation of serum BSP concentration Determination of BSP concentration was carried out according to the method described by Mixner and Robertson21, with the following modifications : I. Because the problem of haemolysis was eliminated, it was possible to make the determination at a single wavelength (580 mp). 2. Colour was developed in “Test” samples by the addition of two drops of 10% NaOH. “Blanks” were prepared by acidification with two drops of 0.25 N HCl. 3. The optical density of the test samples was compared with a standard solution of BSP of 2.5 mg/Ioo ml. A new standard was prepared with each batch of the infusion solution.
Optical densities were read on a Bausch and Lomb Spectronic 20 at 580 rnp with matched, r-cm square cuvettes. BSP concentration was calculated as follows: OD (Test-Blank) BSP (mg/roo ml) = Std. BSP concentration x ~ OD (Std.-Std. Blank) Calculations and statistics Loge of concentration was plotted against time and the line of best fit calculated by the method of least squares. The correlation coefficient (Y) was calculated for each of these lines. The fractional clearance (K) and biological half life (Tg) were determined by Clin. Chim.
Acta,
18
(1967)
1x3-140
HUNT,
136
MCCOSKER
the method outlined by Mixner and Robertson 21.Student’s “t” was calculated to test the difference between the mean “K” values of males, females and steers. An analysis of variance was carried out on the data obtained from the “repeatability” experiment to determine the day-to-day variation within animals. RESULTS
In all 25 animals examined for normal clearance, BSP was excreted from the circulation in such a way that a semi-logarithmic plot of concentration against time resulted in a single straight line. Typical decay curves are illustrated in Fig. I. The detailed results of fractional clearance (K), half life (T$) and correlation coefficient (r) of the lines obtained are given in Tables I, II and III for entire males, entire females and steers respectively. From these it will be seen that the correlation coefficients, with one exception (bovine ao3-Table I), fall between 0.9838 and I.OOOO. This is a TABLE
I
Identifcafmn
Weight (lbs.)
Dose
K
T+ (min)
Y*
3.85 4.08 3.46
0.9998 0.9997 0.9986
(wf)
~.
72 73 74
225 326 3oo
225 32s ,300
0.181 0.148 0.200
94 96 97
247 437 413
250 450 400
0.1gr
99
310
350
0.162
203
20.3
LOO
O.I22
204 208
266 274
250 3oo
203 213
368 375
350 375
MeaIl
‘: S.D.
Identifcation
0.9993 0.9969 0.9979
0.167 0.151
4.42 5.67 4.14 4.57
0.9996 0.9030 0.9838 0.9922
0.099 0.153
7.04 4.52
0.997 1 0.9992
3.85
0.180
_____.
* r = Correlation
4.51 3.86
0.181
0.158
k 0.027
4.39
I: 0.93
coeflicicnt.
Weight (lhs.)
DOS63
K
T-h (vnin)
Y*
Acute I 54 55
3oo 350 338
3oo 350 350
0.126 0.172 0.224
5.50 4.02 3.15
0.995 1 0.9960 1.0000
56 64
354 522
350 5oo
0.193 0.179
3.58 3.88
0.9995 0.995 1
(m&7)
201
362
350
0.136
5.08
0.985s
205 206 207
381 306 313
4oo 300 3oo
0.744 0.242 0.141
4.83 2.87 4.91
0.9993 0.9987 0.9973
Mean
+ S.D.
* r = Correlation C&n. Chim. Acta,
0.173 coefficient. 18 (1967) 133-140
I_ 0.039
4.00
:: 0.86
BSP
CLEARANCE IN CATTLE
I37
very high degree of correlation details,
i.e. elimination
and supports
of haemolysis
the satisfactory
nature
and use of the “Braunula”
of the technical
to obtain accurately
timed blood samples. TABLE
III
FRACTIoNAL CLEARANCE(K) Identifccatmn
88 92 202 212
ANDHALFLIFE (T&) FORFOURYEARLIKGHEREFORD CATTLE-STEERS
Weight (lbs.)
DO.%
255 396 378 322
250 400 375 325
K
TQ (min)
Y*
0.148
4.66
0.9920
0.139 0.202 0.206
4.96 3.40 3.34
0.9978 0.9780 I .oooo
f %)
Mean ‘- S.D.
0.174
* r = Correlation
*
0.097
3.98
1: 0.73
coefficient.
The mean fractional
clearances
of the different
groups of cattle were 0.158 &
0.027, 0.173 & 0.039 and 0.174 + 0.097 for entire males, entire females and steers respectively. Statistical analysis, Student’s “t” test, shows no significant difference between these groups. In order to check the repeatability for BSP fractional clearance Results of these estimations
of the method,
(Table V) it can be seen that the day-to-day TABLE
six animals were examined
on three consecutive days under identical are shown in Table IV. From the analysis variation
conditions. of variance
within animals is not significant.
IV
REPEATABILITYOF FRACTIONALCIXARANCE ESTIMATIONS Identifi-
Day
401 402 403* 404
0.228 0.172 0.216 0.235
0.189 0.174 0.239 0.195
0.189 0.196 0.248 0.191
73 74
0.148 0.200
0.161 0.187
0.147 0.205
cation
K
I
Day K
2
Day K
3
K, and K, are fractional clearance values obtained on three consecutive days. * A biphasic curve was obtained in all three tests on this animal. The K value of the first phase only has been included in this table. K,,
TABLE
V
Anal_vsis
o_f variance
Between animals Within animals
of repeatability
results
(Table IV)
DF
ss
MS
5 12
0.0113 0.0034
0.0026 0.00028
DISCUSSION One of the early technical problems encountered was the difficulty of obtaining accurately timed serial blood samples. Initially, an indwelling plastic cannula was Clin.
Chim.
Acta,
18 (1967)
133-140
HUXT, YCCOSKER
138
inserted into the jugular vein with the aid of a MacGregor needle. This method proved to be unsatisfactory as a haematoma tended to form due to the escape of blood around the cannula when pressure was applied to the jugular vein to facilitate sample collection. The subsequent use of the “Braunula” solved this problem of timed serial samples of blood became simple and accurate. Haemolysis tion of BSP
of blood samples presents
concentration,
because
and collection
a problem in spectrophotometric
the optimum
absorption
wavelength
estimaof BSP
(580 m,u) coincides with that of haemoglobin. Mixner and Robertson2r have outlined a method which overcomes this problem. However, the great disadvantage of this method lies in the fact that estimations and a calculated
optical density
must be made at three different
(COD) estimated.
The method
wavelengths
of collection
here eliminated the problem of haemolysis and made possible accurate of BSP concentration at the one wavelength (580 mp).
outlined
determination
Clearance of BSP from the plasma was examined in 25 yearling betf cattle whose weights ranged from 200 to 522 Ibs. In all cases, the logarithm of dye concentration varied inversely with time and an excellent degree of linearity occurred over 1
_ 8.04
74 CK= 0.185) -v
4
-8
- ~p-~~ 12 Time
Fig, I. BSP fractional tional clearance (K)
II
16
20
24
(mln)
clearance curves for Calves 74, 94, g6 and gg. Figures in brad&s
= frac-
the time interval 4 to 16 min, following intravenous administration of dye at the rate of I mg/lb. body weight. The correlation coefficients (r) which were obtained (Tables I to III) confirm this. In all of the group of 25 animals examined to define the normal fractional clearance, a single exponential line was obtained. However, in a further group of 6 animals examined to test the repeatability of the “K” value, a biphasic curve was Clin. Chin<. Acta,
18
(1967) 133-140
BSP
CLEARANCE
obtained ditions
IN CATTLE
in one animal. outlined,
I39
It would therefore
a single-phase
BSP
appear that in beef cattle under the con-
excretion
pattern
is generally
obtained.
This
agrees with the observations of Mixner and Robertson 21 and Cornelius et aLza in cattle. However, in other domestic species, biphasic curves have been described in the normal animal (Table VI). In contrast to this, Lavers et al. 17 and Goodmanls have shown that a TABLE
VI
BIPHASIC PATTERN OF BSP
EXCRETION
OBSERVED
IN
NORMAL
DOMESTIC
Species
Dose vate
Reference
Dog
5 mg/kg body weight 5 mg/kg body weight 5 mg/kg body weight 2-6 mg/lb. body weight 5-7.5 mg/lb. body weight L g std. dose for average horse
Mills and Dragstedt2’ Cornelius28 Richards et a1.2” Cornelius28 Clarkson Cornelius et a1.23
Dog Dog Sheep Turkey Horse;
* Using a I g standard
biphasic
type curve is obtained
apparently weight)
dose these authors
indicate
in only 25%
that a single-phase
SPECIES
curve is obtained.
of human cases where liver function
is
It is possible that the low dose rate used in cattle (I mg/lb. body as compared with other species (5 mg/kg) and the difficulty experienced in
accurately difference
normal.
detecting low concentrations of BSP, may account for the apparent in excretion pattern. Results obtained by Cornelius et aLz3 on the horse
tend to confirm this. Using a standard dose of I g for the average horse, they obtained a single straight line excretion curve. This dose would approximate to I mg/lb. body weight. Using a standard dose of z g for the average horse, their results indicate that a biphasic curve is obtained. However, it has been shownz4 recently in sheep, that a dose rate of 5 mg/kg body weight resulted in a single-phase pattern of excretion, similar to that which occurs in cattle. This suggests that a different pattern of excretion of BSP might exist in ruminant animals which may be accounted for by some slight physiological difference. The significance of the the biphasic pattern of excretion has been considered in relation to the dogzS. These authors suggest that “The process of hepatic excretion of BSP
involves
a dual mechanism:
(a) prompt removal
from the blood and tempo-
rary storage in liver cells, (b) more gradual subsequent transfer from these cells into the lumens of the bile canaliculi.” This hypothesis is supported by the work of Richards et aLz6 also on the dog. On this basis, the difference in excretion pattern observed in cattle and sheep compared with other species may be due to slight difference in any or all of the following functions: hepatic blood flow, transfer of dye from blood to the liver cell, storage capacity of the liver cells and transfer of dye from liver cells into bile canaliculi. The mean fractional clearance for this group of z5 animals was 0.166 f 0.034. This compares well with the results obtained by Mixner and Robertson21 (K = 0.156 for nine Holstein bull calves) and Cornelius et akz2 (K = 0.155 for five Ia-r&monthold Hereford steers and heifers).
Clin. Chim.
Acta,
18 (1967)
1x3-140
HUNT,
140
MCCOSKER
ACKNOWLEDGMENTS
We wish to acknowledge
the support of the Australian
Meat Research
Committee
for this work, the assistance analysis, and the technical
of Mr. A. W. Beattie and Mrs. P. Burgess with statistical assistance of P. Amos and N. Doyle. We also wish to
acknowledge
and encoul-agelllent
I L 3 4 5 0 7
the assistance
of Professor
D. I;. Dow-ling.
S. 31. ROSENTHAL .XKD 1-I.C. WHITE, J. .4m. &fed. Assoc., 84 (1925) IIIZ. C. MOSES, T. H. CRITCHFIELD AND T. B. THOMAS, I. Lab. C/in. hIed., 3.3 (1948) C. COHN, It. LEVIWE AND iVl. KOLINSKY, .4m. J. Ph>&>l., 155 (1948) 286, 1<. W. BRAVER, I<. L. I'ESSOTI AND J. S. ICREBS, J. Cliz. lwest., 34 (1955) 35 ti. J. L.ARSON ANU C. C. MORRIL, Am. J. Vet. I?es.,21 (1gOo) 94.9. 1. ibf.p\TIELSoN, i\'ovd. C’etevinamzed., 4 (I9j2) 1192. 1% 1;. HOKLEIN ANI) [. E. GREEN. North Am. Fft., 31 (1950) 662.
4~8.
CHIMICA
ACTA
BROMSULPHTHALEIN
I33
FRACTIONAL
CLEARANCE
IN NORMAL
BEEF
CATTLE
S. E. HUNT
AND P. J. McCOSKER
Department of Clinical (Received
June
Igth,
Studies, Veterinavy School, University of Queensland (Australia) * 1967)
SUMMARY
Application of the BSP fractional clearance test to beef cattle, has been studied. A simplified method for the collection of accurately timed serial blood samples free of haemolysis is presented. This involves the use of an indwelling cannula (Braunula). Elimination of haemolysis simplifies spectrophotometric estimation of BSP concentration, as no correction for contamination by haemoglobin is necessary and readings may be made at one wavelength (580 mp). Mean BSP fractional clearance of 25 beef cattle (200-522 0.166 & SD 0.034, with half life (T&) 4.18 min. No significant served. The reproducibility of results using this method analysis of variance of the data shows that the day-to-day is not significant.
lbs body weight) was sex difference was ob-
has been examined and an variation within individuals
The different excretion pattern observed in normal cattle, namely single-phase as compared with biphasic, which occurs in other species, has been discussed.
INTRODUCTION
Rosenthal and White’ developed the original Bromsulphthalein (BSP) liver function test in human medicine. In their test, the percentage of dye remaining in the plasma at 5 and 30 min from the time of intravenous injection of BSP at the rate of 2 mg per kg body weight, was used as an index of liver function. Application of BSP retention as a test of liver function in cattle has not always yielded satisfactory results 10--15.A liver function test based on this method is currently being used as a satisfactory index of liver function in the dog2-7. Drill and Ivy8 compared the BSP retention test with serum alkaline phosphatase, prothrombin time, galactose tolerance and indirect Van den Bergh estimations. They concluded that BSP retention was the most sensitive of these tests. Svirbely et al9 found that the BSP retention test was more sensitive than Rose Bengal retention and more reliable than bilirubin estimation or urinary urobilinogen. * Mailing
addrex:
Mill Road,
St. Lucia,
Queensland. Clin.
Chim.
Acta, 18 (1967)
133-140
HUNT, MCCOSKER
I34
Ingelfinger et al. I6 have shown that, in man, concentration of dye in the plasma after intravenous injection of a single dose, declines exponentially and a straight line is obtained when log, of concentration is plotted against time. This has been confirmed by Lavers et 61.” and Goodmanis, who suggested that the slope of the line so produced would provide a quantitative measure of liver function. This function has been termed “fractional clearance” by Lewisls~zo who defined this term as “the fraction of the total fluid volume (or solvent compartment) cleared in unit time”. Mixner and Robertson2* applied the principle of fractional clearance of BSP to the study of normal liver function in dairy cattle. They found that over the period 3-15 min the results obeyed the exponential law suggested by Ingelfinger et al.16. The mean BSP fractional clearance for nine Holstein bull calves and five lactating Holstein cows was 0.156 and 0.1gz respectively. Cornelius et aLz2 record results of BSP fractional clearance for fifteen non-lactating dairy cows as, 0.22 & 0.03, and for five Iz-r&month-old Hereford steers and heifers as 0.155 & 0.011. Hansenz3 was unable to confirm that BSP is excreted exponentially in cattle. The object of this paper is to present the results obtained for BSP fractional clearance in normal beef cattle. MATERIALS AND METHODS
Animals
Twenty-five normal yearling Hereford or Hereford cross cattle, zoo to 522 lbs. body weight, were examined. They comprised twelve entire males, nine entire females and four steers. They were housed in metabolism cages and a settling in period of one week was allowed before experiments were commenced. Throughout the experimental period, water and lucerne chaff were supplied ad lib. In addition, six similar animals, 300-350 lbs. weight, were examined three times on consecutive days under identical conditions to ascertain the repeatability of the method. All six animals were males, four being Shorthorn-Hereford crosses and two Herefords. Fractional
clearance
technique
The determination of fractional clearance is dependent upon collection of accurately timed blood samples, free of haemolysis or turbidity. The method of collection outlined below, ensured that accurately timed blood samples free of haemolysis were obtained. Further, the variable degree of turbidity which occurred in heparinised plasma samples was eliminated when serum was collected instead. Infusion
and collectiort
of samples
A 5% aqueous solution of BSP* was used. Distilled water was used in preference to physiological saline as it was found that BSP precipitated out of solution in the latter at room temperature. This solution was sterilised by autoclaving. Infusion and collection of samples was facilitated by the introduction of a “Braunula zL”** into the jugular vein. A small area over the jugular vein was shaved * Sulphobromophthalein Sodium salt. Koch-Light Laboratories Ltd. Code No. 51~4h. ** Manufactured by K. Braun, Melsungen, W. Germany. Obtainable in Australia from Boots Pure Drug Co. Ltd. (The “Braunula” comprises three components: an outer plastic cannula, a 34” needle insert which protudes $” beyond the plastic cannula and a plastic plug, the end of which is sealed with a gum rubber cap.) CZin. Chiwz. Acta,
18 (1967) 133-140
BSP
CLEARANCE
and cleaned
I35
IN CATTLE
with 0.1%
Cetavlon.
Local
anaesthetic
(3% Citanest)
was infused
into
the area and a stab incision was made with a scalpel. The “Braunula” was inserted into the jugular vein via the stab incision, and its needle insert withdrawn, leaving the plastic cannula in place in the vein. An initial blood sample was collected by allowing blood to drain into a r5-ml centrifuge tube. The “Braunula” was then sealed with the plastic plug and a few ml of heparinised physiological saline infused through the gum rubber cap to prevent clotting. Infusion of the BSP (I mg/lb. body wt.) was made through the cap. A stopwatch was started at the midpoint of injection. A few ml of heparinised saline were infused in a similar manner to ensure that all the BSP had entered the vein and also to prevent blood clotting in the cannula. This was repeated immediately after each sample collection. Blood samples were collected every 4 min for periods up to 24 min, by removing the plastic plug and allowing blood to flow directly into r5-ml centrifuge tubes, after ensuring that all the heparinised saline in the cannula had been removed.The time was noted at the midpoint of collection. Centr
The estimation of serum BSP concentration Determination of BSP concentration was carried out according to the method described by Mixner and Robertson21, with the following modifications : I. Because the problem of haemolysis was eliminated, it was possible to make the determination at a single wavelength (580 mp). 2. Colour was developed in “Test” samples by the addition of two drops of 10% NaOH. “Blanks” were prepared by acidification with two drops of 0.25 N HCl. 3. The optical density of the test samples was compared with a standard solution of BSP of 2.5 mg/Ioo ml. A new standard was prepared with each batch of the infusion solution.
Optical densities were read on a Bausch and Lomb Spectronic 20 at 580 rnp with matched, r-cm square cuvettes. BSP concentration was calculated as follows: OD (Test-Blank) BSP (mg/roo ml) = Std. BSP concentration x ~ OD (Std.-Std. Blank) Calculations and statistics Loge of concentration was plotted against time and the line of best fit calculated by the method of least squares. The correlation coefficient (Y) was calculated for each of these lines. The fractional clearance (K) and biological half life (Tg) were determined by Clin. Chim.
Acta,
18
(1967)
1x3-140
HUNT,
136
MCCOSKER
the method outlined by Mixner and Robertson 21.Student’s “t” was calculated to test the difference between the mean “K” values of males, females and steers. An analysis of variance was carried out on the data obtained from the “repeatability” experiment to determine the day-to-day variation within animals. RESULTS
In all 25 animals examined for normal clearance, BSP was excreted from the circulation in such a way that a semi-logarithmic plot of concentration against time resulted in a single straight line. Typical decay curves are illustrated in Fig. I. The detailed results of fractional clearance (K), half life (T$) and correlation coefficient (r) of the lines obtained are given in Tables I, II and III for entire males, entire females and steers respectively. From these it will be seen that the correlation coefficients, with one exception (bovine ao3-Table I), fall between 0.9838 and I.OOOO. This is a TABLE
I
Identifcafmn
Weight (lbs.)
Dose
K
T+ (min)
Y*
3.85 4.08 3.46
0.9998 0.9997 0.9986
(wf)
~.
72 73 74
225 326 3oo
225 32s ,300
0.181 0.148 0.200
94 96 97
247 437 413
250 450 400
0.1gr
99
310
350
0.162
203
20.3
LOO
O.I22
204 208
266 274
250 3oo
203 213
368 375
350 375
MeaIl
‘: S.D.
Identifcation
0.9993 0.9969 0.9979
0.167 0.151
4.42 5.67 4.14 4.57
0.9996 0.9030 0.9838 0.9922
0.099 0.153
7.04 4.52
0.997 1 0.9992
3.85
0.180
_____.
* r = Correlation
4.51 3.86
0.181
0.158
k 0.027
4.39
I: 0.93
coeflicicnt.
Weight (lhs.)
DOS63
K
T-h (vnin)
Y*
Acute I 54 55
3oo 350 338
3oo 350 350
0.126 0.172 0.224
5.50 4.02 3.15
0.995 1 0.9960 1.0000
56 64
354 522
350 5oo
0.193 0.179
3.58 3.88
0.9995 0.995 1
(m&7)
201
362
350
0.136
5.08
0.985s
205 206 207
381 306 313
4oo 300 3oo
0.744 0.242 0.141
4.83 2.87 4.91
0.9993 0.9987 0.9973
Mean
+ S.D.
* r = Correlation C&n. Chim. Acta,
0.173 coefficient. 18 (1967) 133-140
I_ 0.039
4.00
:: 0.86
BSP
CLEARANCE IN CATTLE
I37
very high degree of correlation details,
i.e. elimination
and supports
of haemolysis
the satisfactory
nature
and use of the “Braunula”
of the technical
to obtain accurately
timed blood samples. TABLE
III
FRACTIoNAL CLEARANCE(K) Identifccatmn
88 92 202 212
ANDHALFLIFE (T&) FORFOURYEARLIKGHEREFORD CATTLE-STEERS
Weight (lbs.)
DO.%
255 396 378 322
250 400 375 325
K
TQ (min)
Y*
0.148
4.66
0.9920
0.139 0.202 0.206
4.96 3.40 3.34
0.9978 0.9780 I .oooo
f %)
Mean ‘- S.D.
0.174
* r = Correlation
*
0.097
3.98
1: 0.73
coefficient.
The mean fractional
clearances
of the different
groups of cattle were 0.158 &
0.027, 0.173 & 0.039 and 0.174 + 0.097 for entire males, entire females and steers respectively. Statistical analysis, Student’s “t” test, shows no significant difference between these groups. In order to check the repeatability for BSP fractional clearance Results of these estimations
of the method,
(Table V) it can be seen that the day-to-day TABLE
six animals were examined
on three consecutive days under identical are shown in Table IV. From the analysis variation
conditions. of variance
within animals is not significant.
IV
REPEATABILITYOF FRACTIONALCIXARANCE ESTIMATIONS Identifi-
Day
401 402 403* 404
0.228 0.172 0.216 0.235
0.189 0.174 0.239 0.195
0.189 0.196 0.248 0.191
73 74
0.148 0.200
0.161 0.187
0.147 0.205
cation
K
I
Day K
2
Day K
3
K, and K, are fractional clearance values obtained on three consecutive days. * A biphasic curve was obtained in all three tests on this animal. The K value of the first phase only has been included in this table. K,,
TABLE
V
Anal_vsis
o_f variance
Between animals Within animals
of repeatability
results
(Table IV)
DF
ss
MS
5 12
0.0113 0.0034
0.0026 0.00028
DISCUSSION One of the early technical problems encountered was the difficulty of obtaining accurately timed serial blood samples. Initially, an indwelling plastic cannula was Clin.
Chim.
Acta,
18 (1967)
133-140
HUXT, YCCOSKER
138
inserted into the jugular vein with the aid of a MacGregor needle. This method proved to be unsatisfactory as a haematoma tended to form due to the escape of blood around the cannula when pressure was applied to the jugular vein to facilitate sample collection. The subsequent use of the “Braunula” solved this problem of timed serial samples of blood became simple and accurate. Haemolysis tion of BSP
of blood samples presents
concentration,
because
and collection
a problem in spectrophotometric
the optimum
absorption
wavelength
estimaof BSP
(580 m,u) coincides with that of haemoglobin. Mixner and Robertson2r have outlined a method which overcomes this problem. However, the great disadvantage of this method lies in the fact that estimations and a calculated
optical density
must be made at three different
(COD) estimated.
The method
wavelengths
of collection
here eliminated the problem of haemolysis and made possible accurate of BSP concentration at the one wavelength (580 mp).
outlined
determination
Clearance of BSP from the plasma was examined in 25 yearling betf cattle whose weights ranged from 200 to 522 Ibs. In all cases, the logarithm of dye concentration varied inversely with time and an excellent degree of linearity occurred over 1
_ 8.04
74 CK= 0.185) -v
4
-8
- ~p-~~ 12 Time
Fig, I. BSP fractional tional clearance (K)
II
16
20
24
(mln)
clearance curves for Calves 74, 94, g6 and gg. Figures in brad&s
= frac-
the time interval 4 to 16 min, following intravenous administration of dye at the rate of I mg/lb. body weight. The correlation coefficients (r) which were obtained (Tables I to III) confirm this. In all of the group of 25 animals examined to define the normal fractional clearance, a single exponential line was obtained. However, in a further group of 6 animals examined to test the repeatability of the “K” value, a biphasic curve was Clin. Chin<. Acta,
18
(1967) 133-140
BSP
CLEARANCE
obtained ditions
IN CATTLE
in one animal. outlined,
I39
It would therefore
a single-phase
BSP
appear that in beef cattle under the con-
excretion
pattern
is generally
obtained.
This
agrees with the observations of Mixner and Robertson 21 and Cornelius et aLza in cattle. However, in other domestic species, biphasic curves have been described in the normal animal (Table VI). In contrast to this, Lavers et al. 17 and Goodmanls have shown that a TABLE
VI
BIPHASIC PATTERN OF BSP
EXCRETION
OBSERVED
IN
NORMAL
DOMESTIC
Species
Dose vate
Reference
Dog
5 mg/kg body weight 5 mg/kg body weight 5 mg/kg body weight 2-6 mg/lb. body weight 5-7.5 mg/lb. body weight L g std. dose for average horse
Mills and Dragstedt2’ Cornelius28 Richards et a1.2” Cornelius28 Clarkson Cornelius et a1.23
Dog Dog Sheep Turkey Horse;
* Using a I g standard
biphasic
type curve is obtained
apparently weight)
dose these authors
indicate
in only 25%
that a single-phase
SPECIES
curve is obtained.
of human cases where liver function
is
It is possible that the low dose rate used in cattle (I mg/lb. body as compared with other species (5 mg/kg) and the difficulty experienced in
accurately difference
normal.
detecting low concentrations of BSP, may account for the apparent in excretion pattern. Results obtained by Cornelius et aLz3 on the horse
tend to confirm this. Using a standard dose of I g for the average horse, they obtained a single straight line excretion curve. This dose would approximate to I mg/lb. body weight. Using a standard dose of z g for the average horse, their results indicate that a biphasic curve is obtained. However, it has been shownz4 recently in sheep, that a dose rate of 5 mg/kg body weight resulted in a single-phase pattern of excretion, similar to that which occurs in cattle. This suggests that a different pattern of excretion of BSP might exist in ruminant animals which may be accounted for by some slight physiological difference. The significance of the the biphasic pattern of excretion has been considered in relation to the dogzS. These authors suggest that “The process of hepatic excretion of BSP
involves
a dual mechanism:
(a) prompt removal
from the blood and tempo-
rary storage in liver cells, (b) more gradual subsequent transfer from these cells into the lumens of the bile canaliculi.” This hypothesis is supported by the work of Richards et aLz6 also on the dog. On this basis, the difference in excretion pattern observed in cattle and sheep compared with other species may be due to slight difference in any or all of the following functions: hepatic blood flow, transfer of dye from blood to the liver cell, storage capacity of the liver cells and transfer of dye from liver cells into bile canaliculi. The mean fractional clearance for this group of z5 animals was 0.166 f 0.034. This compares well with the results obtained by Mixner and Robertson21 (K = 0.156 for nine Holstein bull calves) and Cornelius et akz2 (K = 0.155 for five Ia-r&monthold Hereford steers and heifers).
Clin. Chim.
Acta,
18 (1967)
1x3-140
HUNT,
140
MCCOSKER
ACKNOWLEDGMENTS
We wish to acknowledge
the support of the Australian
Meat Research
Committee
for this work, the assistance analysis, and the technical
of Mr. A. W. Beattie and Mrs. P. Burgess with statistical assistance of P. Amos and N. Doyle. We also wish to
acknowledge
and encoul-agelllent
I L 3 4 5 0 7
the assistance
of Professor
D. I;. Dow-ling.
S. 31. ROSENTHAL .XKD 1-I.C. WHITE, J. .4m. &fed. Assoc., 84 (1925) IIIZ. C. MOSES, T. H. CRITCHFIELD AND T. B. THOMAS, I. Lab. C/in. hIed., 3.3 (1948) C. COHN, It. LEVIWE AND iVl. KOLINSKY, .4m. J. Ph>&>l., 155 (1948) 286, 1<. W. BRAVER, I<. L. I'ESSOTI AND J. S. ICREBS, J. Cliz. lwest., 34 (1955) 35 ti. J. L.ARSON ANU C. C. MORRIL, Am. J. Vet. I?es.,21 (1gOo) 94.9. 1. ibf.p\TIELSoN, i\'ovd. C’etevinamzed., 4 (I9j2) 1192. 1% 1;. HOKLEIN ANI) [. E. GREEN. North Am. Fft., 31 (1950) 662.
4~8.