Life Sciences, Vol . 23, pp . 2339-2348 Printed in the U .S .A .
Pergamon Press
CONTRIBUTION OF PLASMA HOMOVANILLIC ACID (HVA) TO URINE AND CEREBROSPINAL GLUID HVA IN THE MONKEY AND TTS PHARMACORINETIC DISPOSITION M .A . Elchisak, R .J . Poliaeky, M .H . Ebert, R .J . Powers aad I .J . Ropin Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland 20014 (Received in final form October 16, 1978) SIß~II~lARY Homovanillic acid (HVA) labelled with two deuterium stoma (d 2HVA) was used to determine the contribution of HVA in the blood to HVA in the urine and CSF of monkeys . During and after a sixhour intravenous infusion of d2HVA at a constant rate, the levels of both d2 -HVA and endogenous HVA (dam HVA) in plasma, urine, and CSF were determined by gas chromatography-mesa spectrometry, and the relative enrichments of d~ H~~VA is each of these fluids calculated . Results indicate that -HVA in the urine is derived exclueivelq from the blood, with no contribution from renal metabolism of dopamine (DA) . Furthermore, less than one percent of HVA is either lumbar or ventricular CSF is derived from circulating HVA . The plasma elimination curve of d 2 -riVA was bieaponential, with a terminal phase half-life (t~) of 44 minutes and an apparent volume of distribution of 0 .8 liters/kg . Urinary excretion of homovanillic acid (HVA), the major metabolite of dopamine (DA) in man (1), has been reported to be altered in several disorders, including neuroblastoma (2,3,4), malignant pheochromocytoma (3), acute myo cardial infarction (5), hypertension (6), Parkinson's disease (7,8,9), and other movement disorders (9) . The disturbance of metabolism of HVA is many of these disorders remains unclear . DA, as well as HVA, is present in the urine, but it has been suggested that urinary DA may be of renal, rather than circulatory, origin (6,10,11,12,13) . The kidney is known to contain DA (14), as well ae monoamine oxidase and catechol-0-methyltransferase (15), the enzymes which convert DA to HVA . HVA in urine may therefore be derived from renal DA, se well as from plasma HVA, which is a metabolite of DA in the brain and peripheral tissues . In the present study, the contribution of the 1C1.dney to urinary levels of HVA has been examined . HVA labelled with two deuterium atoms (d 2HVA) was iafuaed into monkeys until a steady-state level was attained . The relative en richment of d 2 -HVA in the plasma and urine was determined by gas chromatography-mass spectrometry (GC-MS) . A direct renal contribution of HVA to the urine would be indicated by a lower relative enrichment of urinary d y -HVA than of plasma d 2 -HVA ; otherwise, the urinary HVA must be derived solely from HVA is the plasma . Additionally, since the peripheral contribution of HVA to the CSF has been reported to be as high as 20 percent (16), the relative enrichment of 0300-9653/78/1204-233902 .00/0
2340
HVA in Monkey Plasma, Urine and CSF
Vol . 23, No . 23, 1978
d2-HVA in lumbar or ventricular CSF was also determined and compared to that in the plasma . METHODS Sub ects : Male rhesus monkeys (macaca mulatta), weighing 5-7 kg were adapted to primate restraining chairs for at least one week prior to the experiment . Stainless steel canaulae were implanted stereotactically sad chronically maintained in the lateral ventricle as previously described (17) . Several days Lumbar catheters were used to collect CSF from the thecal sac . after recovery from surgery, and approximately 18 hours prior to the beginning of the infusion, the animal was lightly anesthetized with ketamine HC1 and xylaziae (7 mg and 0 .6 mg/kg, respectively, i.m .) . A catheter was inserted into each eaphenous vein ; one for infusion of d -HVA and the other for collection of blood samples . A catheter was also insérted into the urinary bladder . During the night before the experiment, five percent dextrose in water (5X D/W, Abbot Laboratories) was slowly infused intravenously at a rate of 4 ml/kg/hr . Between 9 :00 and 10 :00 AM the next morning, blood and urine samples were collected for determination of baseline levels of endogenous HVA (dpHVA) . D2HVA, dissolved in 5X D/W, was then infused . In two preliminary eaperiments, the initial rate of d2-HVA infusion was high (35 .5 ug/kg/hr) for 33 minutes, and then immediately followed by a lower rate of d2-HVA infusion (7 .1 or 14 .2 ug/kg/hr) from 33 to 360 minutes . It was subaequenEly found that the initial loading was unnecessary, and all subsequent animals were infused at rates of 7 .1 or 14 .2 yg d HVA/kg/hr for 360 minutes . Blood samples were collected in heparinized tubeâ, and urine was collected on ice, at appropriate intervals during the course of the infusion, and in one case, for several hours after the infusion was stopped . All samples were frozen and assayed for d~HVA and d2-HVA within two days . Chemical Analysis : The levels of d0-HVA and d2AVA in urine and plasma were determined by gee chromatography-mesa spectrometry (GC-MS) according to methods described elsewhere (J . Oliver, E. Gordon and I. Kopin, in preparation) . D5-HVA was used as internal standard . The ratio of d0-HVA or d2-HVA to d5-HVA in each sample was determined from the peak heights, and the amount of d~ HVA or d -HVA calculated by inverse linear regression analysis of a standard curve cons~ructed for plasma or urine by addition of known amounts of d~HVA to d5-HVA and carried through the extraction procedure . All peak heights were corrected for background and normalized by a program designed by Jendea et al . (18) to correct for varying amounts of channel spillover and natural abundance of the isotopes measured . Calculations : The relative enrichments (RE) of d2-HVA in all samples were calculated according to the formula: [d 2-HVA] d0-HVA
+
d2-HVA
The experimentally-determined RE values were fitted to equation 3 below, using the nonlinear, least-squares computer program, MLAB (19) . The urine or plasma steady-state d -HVA relative enrichment values (RE ) were then obss tained from equation 24, described below. The maximum RE value in ventricular or lumbar CSF was used to calculate the proportion of HVA in CSF derived from the blood according to the following equation : maximum RE in CSF X HVA in CSF derived from blood = % 100 (1) RE ss in plasma
Vol,
23, No . 23, 1978
HVA in Monkey Plasma, Urine and CSF
2341
The experimentally-determined d2-HVA plasma concentrations during the poet-infusion phase were fitted to equation 5 below using MLAB, and various pharmacokinetic parameters thereby obtained (see Reaulte), Equations : Graphical analysis of the changes in plasma level of d2-HVA with time after intravenous administration of a bolus of the labelled compound indicated that the data were best described by a two-exponential function (20) : C~Ae
-at
e
+B
ßt
(2)
This equation implies that IiVA kinetics is best described by a twocompartment body model . The concentration of drug in the plasma at time t is C ; a and ß are the disposition constants of IiVA ; and A and B are the zero-time intercepts obtained by extrapolation to time t ~ 0 of the first linear phase, obtained by the method of residuals, and the terminal linear phase, respectively . When a substance is infused at a constant rate into the central compartment, equation 1 for this model, after rearrangement and simplification, becomes : ß a C ~ k0 10 ~at + k10 ~ßt ~ (3) a-ß a-ß Vc k10
C1
+ -k
where k0 is the infusion rate, V c is the apparent volume of the central compartment, and k10~ related to a and ß .
k21 are _and elimination and transfer rate constants k12' _ at ßt Since e and e approach zero as time approaches
infinity, the steady-state level of drug (Cs ) in urine and plasma is s Css
~
k
0
(20) : (4)
Vc k10
The time course of drug is the plasma after cessation of infusion is described by (20) : C - A'
e
at'
+ B'
e-ßt'
where t' is poet-infusion time and A' and B' are analogous to A and B in equation 2 . A and B are related to A' and B' by the following equations (20) : A ~
B
A' XO a k0 (1-e
-at
)
(6)
B' %0 ß k0(1-~ßt )
where XO is the administered dose and equals the product of the infusion rate and the infusion time, that is, kO T. Materials : a,a,d2-HVA was obtained commercially (Merck, Sharp, ~ Dohme, Canada, Ltd .) and dissolved in normal saline in a concentration of 0.1 mg/ml, passed through a bacterial filter and stored frozen is sterile and pyrogenfree vials until used . Lindstrom et al . (21) .
D5-HVA was prepared according to the method of
Vol . 23, No . 23, 1978
HVA in Monkey 'lasma, Urine and CSF
2342
RESULTS AND DISCUSSION During its infusion at a constant rate, the was of Plasma and Urine : relative enrichment (RE of d2HVA in both the plasma and urine approached the same constant level within the first two hours (Figure 1) . A direct renal contribution to the urinary HVA would reduce the RE of d -IiVA in urine to a level lower than that in plasma, since endogenous HVA for 2med in the kidney would increase the denominator (d~-tiVA) of thQ equation used to calculate RE (see Methods) . Since RE of HVA in urine and plasma were equal, it is evident that the kidney does not contribute significant amounts of IiVA to the urinary HVA .
1
6iD
1>t-HVA INFUSéD~ 11 .2 UC/KCMt f0-3i0 AIN) 0 UtINE X PLA3AA
.9 Â
.8
x N .3
0
60
120
180
240
300
360
A1N AFTER START ~ iPFU8i0N FIG . 1 Relative enrichment of d2 HVA in monkey plasma and urine during a constant rate d2 äVA infusion ae described in the text . In three cases, The results of five experiments are summarized in Table 1 . the RE of d2-HVA at steady-state (REss ) in the urine was equal to REss in the in urine was plasma . In the two monkeys given the low dose infusion, RE greater than RE in plasma . This effect was probably due sa to an experimental artifact in thesâssay procedure related to the low dose of d2-HVA and low RE, since it was not seen when one of these animals (~68D) received the higher dose . These reaulte inIa no experiment was RE in urine less than RE in plasma . dicate that urinary HVÂis derived solely from splasma I3VA, with no FIVA derived These results are surprising, since much DA in from renal metabolism of DA . the urine is attributed to kidney release of DA (6,10,11,12,13), and it might be expected that the amine would readily be metabolized to HVA.
HVA in Monkey Plasma, Urine and CSF
Vol . 23, No . 23, 1978
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2343
2344
Vol . 23, No . 23, 1978
HVA in Monkey Plasma, Urine and CSF
RE of CSF : The results shown in Table 1 also indicate that leas than one percent of the HVA found in lumbar or lateral ventricular CSF is derived from the circulating HVA . Thin is a maximal figure, since the point chosen for calculation from equation 1 was the mazimima RE observed at any time in the CSF . Figure 2 shown the RE of d -HVA in the ventricular CSF of monkey #68D at various times after begianing2 the infusion . The RE of d2 -HVA appeared to peak 12 hours after beginning the six-hour infusion . Similar results were seen in the other monkey with a ventricular canaula, ~118D (Table 1) . Although the effect is slight, each peak was at least 6 standard deviations above the 0 to 12 hour mean . D -HVA appeared in lumbar CSF during the course of the infusion (Table 1, #79D), iâdicating that this is probably a direct exchange between the blood and lumbar CSF or development of a small leakage of plasma into CSF, although there was no evidence of blood in the CSF . The delayed appearance of d2 HVA in ventricular CSF suggests a mechanism other than direct transfer .
68D
VENTRICULAR CSF D2-FNA IPFUSED~
11 .2 UC/KC/HR,
0
3
0 TO 6 HR
6 9 12 15 18 HOURS AFTER START OF INFUSION
21
FIG. 2 Relative enrichment of d2 -HVA in monkey CSF during and after a constant rate d2 -HVA infusion as described in the teat . These results indicate that HVA in monkey CSF originates almost wholly Except for one study in anesthetized cats in which CSF was obfrom brain. tained by occipital puncture (16), similar results using other techniques have been obtained in other species, including rat (22), cat (23), dog (24), and human (25) . When high doses of HVA are given, however, aubstantlal penetration from the blood into the CSF may occur (26), but wader physiological conditions this transfer appears to be insignificant .
HVA in Monkey Plasma, Urine and CSF
Vol . 23, No . 23, 1978
2345
100 6~D DZ-FNAINFUÔëD~ -_ 11 .t UQ/KG/Fft (0-3f60 A1N1
C C
C L
0
20
~0-
60
80
100
120
1~0
MIN AFTER 0~ OF INFUSIp~I
FIG . 3 Time-course of d -HVA in monkey plasma after cessation of constant rate d 2 -HVA infusion as deécribed in the teat . Each point was determined in duplicate . TABLE 2 Pharmacokinetic Parameters for d2 -HVA Disposition in Monkey #6ßD 1 Parameter
Method of Calculation 2
A' B' a ß
Obtained from MLAB fit of data to equation 5 (see Methods)
A B
Equation 6 (see Methods) Equation 7 (see Methods)
t~(a) 3 t~s(ß) 3 v~4 x
0 .693/a O .693/ß o /(A + B)
Calculated Value 16 .2 ng/ml 6 .28 ng/ml-1 .063 min . -1 .016 min . 366 ng/ml 35 .6 ng/ml 11 .0 min . 44 .2 min . .256 1/kg
1 A11 parameters obtained from poet-infusion phase, after 14 .2 ug/kg/hr d2HVA for six hours . Body weight was 5 .0 kg . 2 A11 equations obtained from Gibaldi and Perrier (20) 3 Half-life of initial and terminal phases, respectively `'Volume of distribution of the central compartment S Apparent volume of distribution
2346
HVA in Monkey Plasma, Urine and CSF
Pharmacokinetic Parametern of d2-HVA :
Vol . 23, No . 23, 1978
The time course of the decline
of d2-HVA in the plasma after cesnatioa of infusion ie shown in Figure 3. Since a semi-logarithmic plot of d2-HVA concentration in plasma versus time is clearly biexponential, a two-compartment model was assumed to dencribe these data . Table 2 shows the disposition parameters calculated from these data . The half-life of the terminal phase [tß(ß)] ie 44 .2 minutes, which is in good agreement with that found in man after a bolus injection of d2-HVA by Anggard et al . (27) . Even though these investigators ignored the initial rapid phase of HVA distribution, the apparent volume of distribution (VB) of d2-HVA is this monkey of 0 .813 1/kg is reasonably close to the mean of 0.59 1/kg in man (27) . The distribution of d -HVA in both man and monkey after intravenous bolus injection is probably beet 2described by a three-exponential function, if the initial distribution phase is seen with rapid early sampling (unpublished observations) . Experiments are currently in progress to assess the relative importance of each compartment to the total body turnover . CONCLUSIONS HVA in the urine is derived from HVA in the blood and not directly from renal metabolism of DA . Less than one percent of HVA in either lumbar or ventricular CSF appears to be derived from the blood . The disposition of plasma HVA in the monkey fits at least a two-compartment model, assuming that the kinetics of HVA are not altered with enlargement of the body pool of HVA by infusion of d2-HVA . ACKNOWLEDGEMENTS We thank Mr . Lyle Modlin for expert technical assistance and Mr . Walter Chmelewski and Mn . Melody Herbat for excellent assistance with the chemical assays . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
T . NAGATSU, Biochemistry of Catecholamines , p . 260, University Park Press, Baltimore (1973) . L . HELSON, M. FLEISHER, V . BETRUNE, M .L . MURPHY, and M.K . SCHWARTZ, Clin . Chem . _18 613-615 (1972) . S . IMASHUKU, H . TARADA, T . SAWADA, T . NAKAMURA, and E .H . LaBROSSE, Cancer _36 450-457 (1975) . M.A . BREWSTER, D .H . BERRY, and M. MORIARTY, Clin . Chew . _23 2247-2249 (1977) . W . JANUSZEWICZ, M . SZNAJDERMAN, B . WOCIAL, J . PREIBISZ, and W. POPLAWSKA, Clin . Chim . Acts . _32 261-264 (1971) . J .L . CUCHE, 0 . KUCHEL, A. BARBEAU, and J . GENEST, Can. Med . Aseoc. J . _22 443-446 (1975) . U .K . RINNE, V . SONNINEN, and J . PALO, Paychiat, et Neurol . (Basel) _151 321-327 (1966) . A. BARBEAU, Agressologie _9 195-200 (1968) . J .A .R . LENMAN, M .J . TURNBULL, A . REID, and A.M . FLEMING, J, Neurol . Sci . _32 219-225 (1977) . U.S . VON EDLER, I . FLOODING, and F . ISCHAJRO, Ac ta . Soc. Med . U~nal . _64 217-225 (1959) . R .W . ALEXANDER, J .R . GILL, Jr ., H. YAMABE, W. LOVENBERG, and H.R, KEISER, J . Clin . Invest . _54 194-200 (1974) . 0 . KUCHEL, J .L . CUCHE, N .T . BW, and J. GENEST, Am . J. Med . Sci . _272 263-268 (1976) . B . FAUCHEUX, N .T . BUD, and 0 . KUCAEL, Am . J. Phyaiol . 232 F123-F127 (1977) .
Val . 23, No . 23, 1978
14 . 15 . 16 . 17 . 18 . 19 . 20 . 21, 22, 23 . 24 . 25 . 26, 27 .
in Monkey Plasma, Urine and CSF
2347
L .I . GOLDBERG, Pharmacol, Rev . 24 1-24 (1972) . M . SANDLER and C .R .J . RUTHVEN, Px~ress in Medicinal Chemistry , vol . b, pp , 200-265, Plen~ Press, New York (1959} . G . BARTHOLINI, A . PLETSCH$R, and R . TISSOT, Ex_perientia 22 609-610 (1966) . E . GORDON, M . PERId)W, J . OLIVER, M . EBERT, and I . KOPIN, Neurochem. 2S 347-349 f197S} . D .J . JENDEN, M, ROCH, and R,A, BOOTH, Anal . Biochem, SS 438-448 (1973} . G .D . KNOTT and D .R, REECE, Proceedin a of the ONLINE X 72 International Conference , vol, 1, pg . 497-526, Brunel University, England 1972 . M . GIBALDI and D . PERRIER, Pharmacokinetics , pp . 45-80, Marcel Dekker, Inc ., New York {1975) . B . LINDSTROM, B, S30QUIST, and E . ANGGARD, J . Labelled Comp . 10 187-194 (1974) . M .L . AIEENSTEIN, and J . KORF, Brain Res , 1 49 129-140 (1978) . M. BULAT, M . JAKÜPCEVZC, and 2 . LACKOVIC,Neurosciences Abstracts 4 156 (1974} . H .C . GOLDBERG, and C,M . YATES, Brat . J . Pharmscol . 33 457-471 (196$} . A. PLETSCHER, G, ~ARTHOLINI, and R . TISSOT, Brain Res , _4 106-109, (1967} . L, PROCKOP, S . FAHN, and P . BARHOUR, Brais Res , 80 435-442 (1974} . E . ANGGARD, T, LEWANDER, and B . SJOQUZST, Life Sci . 1$ 111-122 (1974) .
J.
Pergamon Press
CONTRIBUTION OF PLASMA HOMOVANILLIC ACID (HVA) TO URINE AND CEREBROSPINAL GLUID HVA IN THE MONKEY AND TTS PHARMACORINETIC DISPOSITION M .A . Elchisak, R .J . Poliaeky, M .H . Ebert, R .J . Powers aad I .J . Ropin Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland 20014 (Received in final form October 16, 1978) SIß~II~lARY Homovanillic acid (HVA) labelled with two deuterium stoma (d 2HVA) was used to determine the contribution of HVA in the blood to HVA in the urine and CSF of monkeys . During and after a sixhour intravenous infusion of d2HVA at a constant rate, the levels of both d2 -HVA and endogenous HVA (dam HVA) in plasma, urine, and CSF were determined by gas chromatography-mesa spectrometry, and the relative enrichments of d~ H~~VA is each of these fluids calculated . Results indicate that -HVA in the urine is derived exclueivelq from the blood, with no contribution from renal metabolism of dopamine (DA) . Furthermore, less than one percent of HVA is either lumbar or ventricular CSF is derived from circulating HVA . The plasma elimination curve of d 2 -riVA was bieaponential, with a terminal phase half-life (t~) of 44 minutes and an apparent volume of distribution of 0 .8 liters/kg . Urinary excretion of homovanillic acid (HVA), the major metabolite of dopamine (DA) in man (1), has been reported to be altered in several disorders, including neuroblastoma (2,3,4), malignant pheochromocytoma (3), acute myo cardial infarction (5), hypertension (6), Parkinson's disease (7,8,9), and other movement disorders (9) . The disturbance of metabolism of HVA is many of these disorders remains unclear . DA, as well as HVA, is present in the urine, but it has been suggested that urinary DA may be of renal, rather than circulatory, origin (6,10,11,12,13) . The kidney is known to contain DA (14), as well ae monoamine oxidase and catechol-0-methyltransferase (15), the enzymes which convert DA to HVA . HVA in urine may therefore be derived from renal DA, se well as from plasma HVA, which is a metabolite of DA in the brain and peripheral tissues . In the present study, the contribution of the 1C1.dney to urinary levels of HVA has been examined . HVA labelled with two deuterium atoms (d 2HVA) was iafuaed into monkeys until a steady-state level was attained . The relative en richment of d 2 -HVA in the plasma and urine was determined by gas chromatography-mass spectrometry (GC-MS) . A direct renal contribution of HVA to the urine would be indicated by a lower relative enrichment of urinary d y -HVA than of plasma d 2 -HVA ; otherwise, the urinary HVA must be derived solely from HVA is the plasma . Additionally, since the peripheral contribution of HVA to the CSF has been reported to be as high as 20 percent (16), the relative enrichment of 0300-9653/78/1204-233902 .00/0
2340
HVA in Monkey Plasma, Urine and CSF
Vol . 23, No . 23, 1978
d2-HVA in lumbar or ventricular CSF was also determined and compared to that in the plasma . METHODS Sub ects : Male rhesus monkeys (macaca mulatta), weighing 5-7 kg were adapted to primate restraining chairs for at least one week prior to the experiment . Stainless steel canaulae were implanted stereotactically sad chronically maintained in the lateral ventricle as previously described (17) . Several days Lumbar catheters were used to collect CSF from the thecal sac . after recovery from surgery, and approximately 18 hours prior to the beginning of the infusion, the animal was lightly anesthetized with ketamine HC1 and xylaziae (7 mg and 0 .6 mg/kg, respectively, i.m .) . A catheter was inserted into each eaphenous vein ; one for infusion of d -HVA and the other for collection of blood samples . A catheter was also insérted into the urinary bladder . During the night before the experiment, five percent dextrose in water (5X D/W, Abbot Laboratories) was slowly infused intravenously at a rate of 4 ml/kg/hr . Between 9 :00 and 10 :00 AM the next morning, blood and urine samples were collected for determination of baseline levels of endogenous HVA (dpHVA) . D2HVA, dissolved in 5X D/W, was then infused . In two preliminary eaperiments, the initial rate of d2-HVA infusion was high (35 .5 ug/kg/hr) for 33 minutes, and then immediately followed by a lower rate of d2-HVA infusion (7 .1 or 14 .2 ug/kg/hr) from 33 to 360 minutes . It was subaequenEly found that the initial loading was unnecessary, and all subsequent animals were infused at rates of 7 .1 or 14 .2 yg d HVA/kg/hr for 360 minutes . Blood samples were collected in heparinized tubeâ, and urine was collected on ice, at appropriate intervals during the course of the infusion, and in one case, for several hours after the infusion was stopped . All samples were frozen and assayed for d~HVA and d2-HVA within two days . Chemical Analysis : The levels of d0-HVA and d2AVA in urine and plasma were determined by gee chromatography-mesa spectrometry (GC-MS) according to methods described elsewhere (J . Oliver, E. Gordon and I. Kopin, in preparation) . D5-HVA was used as internal standard . The ratio of d0-HVA or d2-HVA to d5-HVA in each sample was determined from the peak heights, and the amount of d~ HVA or d -HVA calculated by inverse linear regression analysis of a standard curve cons~ructed for plasma or urine by addition of known amounts of d~HVA to d5-HVA and carried through the extraction procedure . All peak heights were corrected for background and normalized by a program designed by Jendea et al . (18) to correct for varying amounts of channel spillover and natural abundance of the isotopes measured . Calculations : The relative enrichments (RE) of d2-HVA in all samples were calculated according to the formula: [d 2-HVA] d0-HVA
+
d2-HVA
The experimentally-determined RE values were fitted to equation 3 below, using the nonlinear, least-squares computer program, MLAB (19) . The urine or plasma steady-state d -HVA relative enrichment values (RE ) were then obss tained from equation 24, described below. The maximum RE value in ventricular or lumbar CSF was used to calculate the proportion of HVA in CSF derived from the blood according to the following equation : maximum RE in CSF X HVA in CSF derived from blood = % 100 (1) RE ss in plasma
Vol,
23, No . 23, 1978
HVA in Monkey Plasma, Urine and CSF
2341
The experimentally-determined d2-HVA plasma concentrations during the poet-infusion phase were fitted to equation 5 below using MLAB, and various pharmacokinetic parameters thereby obtained (see Reaulte), Equations : Graphical analysis of the changes in plasma level of d2-HVA with time after intravenous administration of a bolus of the labelled compound indicated that the data were best described by a two-exponential function (20) : C~Ae
-at
e
+B
ßt
(2)
This equation implies that IiVA kinetics is best described by a twocompartment body model . The concentration of drug in the plasma at time t is C ; a and ß are the disposition constants of IiVA ; and A and B are the zero-time intercepts obtained by extrapolation to time t ~ 0 of the first linear phase, obtained by the method of residuals, and the terminal linear phase, respectively . When a substance is infused at a constant rate into the central compartment, equation 1 for this model, after rearrangement and simplification, becomes : ß a C ~ k0 10 ~at + k10 ~ßt ~ (3) a-ß a-ß Vc k10
C1
+ -k
where k0 is the infusion rate, V c is the apparent volume of the central compartment, and k10~ related to a and ß .
k21 are _and elimination and transfer rate constants k12' _ at ßt Since e and e approach zero as time approaches
infinity, the steady-state level of drug (Cs ) in urine and plasma is s Css
~
k
0
(20) : (4)
Vc k10
The time course of drug is the plasma after cessation of infusion is described by (20) : C - A'
e
at'
+ B'
e-ßt'
where t' is poet-infusion time and A' and B' are analogous to A and B in equation 2 . A and B are related to A' and B' by the following equations (20) : A ~
B
A' XO a k0 (1-e
-at
)
(6)
B' %0 ß k0(1-~ßt )
where XO is the administered dose and equals the product of the infusion rate and the infusion time, that is, kO T. Materials : a,a,d2-HVA was obtained commercially (Merck, Sharp, ~ Dohme, Canada, Ltd .) and dissolved in normal saline in a concentration of 0.1 mg/ml, passed through a bacterial filter and stored frozen is sterile and pyrogenfree vials until used . Lindstrom et al . (21) .
D5-HVA was prepared according to the method of
Vol . 23, No . 23, 1978
HVA in Monkey 'lasma, Urine and CSF
2342
RESULTS AND DISCUSSION During its infusion at a constant rate, the was of Plasma and Urine : relative enrichment (RE of d2HVA in both the plasma and urine approached the same constant level within the first two hours (Figure 1) . A direct renal contribution to the urinary HVA would reduce the RE of d -IiVA in urine to a level lower than that in plasma, since endogenous HVA for 2med in the kidney would increase the denominator (d~-tiVA) of thQ equation used to calculate RE (see Methods) . Since RE of HVA in urine and plasma were equal, it is evident that the kidney does not contribute significant amounts of IiVA to the urinary HVA .
1
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180
240
300
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A1N AFTER START ~ iPFU8i0N FIG . 1 Relative enrichment of d2 HVA in monkey plasma and urine during a constant rate d2 äVA infusion ae described in the text . In three cases, The results of five experiments are summarized in Table 1 . the RE of d2-HVA at steady-state (REss ) in the urine was equal to REss in the in urine was plasma . In the two monkeys given the low dose infusion, RE greater than RE in plasma . This effect was probably due sa to an experimental artifact in thesâssay procedure related to the low dose of d2-HVA and low RE, since it was not seen when one of these animals (~68D) received the higher dose . These reaulte inIa no experiment was RE in urine less than RE in plasma . dicate that urinary HVÂis derived solely from splasma I3VA, with no FIVA derived These results are surprising, since much DA in from renal metabolism of DA . the urine is attributed to kidney release of DA (6,10,11,12,13), and it might be expected that the amine would readily be metabolized to HVA.
HVA in Monkey Plasma, Urine and CSF
Vol . 23, No . 23, 1978
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2343
2344
Vol . 23, No . 23, 1978
HVA in Monkey Plasma, Urine and CSF
RE of CSF : The results shown in Table 1 also indicate that leas than one percent of the HVA found in lumbar or lateral ventricular CSF is derived from the circulating HVA . Thin is a maximal figure, since the point chosen for calculation from equation 1 was the mazimima RE observed at any time in the CSF . Figure 2 shown the RE of d -HVA in the ventricular CSF of monkey #68D at various times after begianing2 the infusion . The RE of d2 -HVA appeared to peak 12 hours after beginning the six-hour infusion . Similar results were seen in the other monkey with a ventricular canaula, ~118D (Table 1) . Although the effect is slight, each peak was at least 6 standard deviations above the 0 to 12 hour mean . D -HVA appeared in lumbar CSF during the course of the infusion (Table 1, #79D), iâdicating that this is probably a direct exchange between the blood and lumbar CSF or development of a small leakage of plasma into CSF, although there was no evidence of blood in the CSF . The delayed appearance of d2 HVA in ventricular CSF suggests a mechanism other than direct transfer .
68D
VENTRICULAR CSF D2-FNA IPFUSED~
11 .2 UC/KC/HR,
0
3
0 TO 6 HR
6 9 12 15 18 HOURS AFTER START OF INFUSION
21
FIG. 2 Relative enrichment of d2 -HVA in monkey CSF during and after a constant rate d2 -HVA infusion as described in the teat . These results indicate that HVA in monkey CSF originates almost wholly Except for one study in anesthetized cats in which CSF was obfrom brain. tained by occipital puncture (16), similar results using other techniques have been obtained in other species, including rat (22), cat (23), dog (24), and human (25) . When high doses of HVA are given, however, aubstantlal penetration from the blood into the CSF may occur (26), but wader physiological conditions this transfer appears to be insignificant .
HVA in Monkey Plasma, Urine and CSF
Vol . 23, No . 23, 1978
2345
100 6~D DZ-FNAINFUÔëD~ -_ 11 .t UQ/KG/Fft (0-3f60 A1N1
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0
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80
100
120
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FIG . 3 Time-course of d -HVA in monkey plasma after cessation of constant rate d 2 -HVA infusion as deécribed in the teat . Each point was determined in duplicate . TABLE 2 Pharmacokinetic Parameters for d2 -HVA Disposition in Monkey #6ßD 1 Parameter
Method of Calculation 2
A' B' a ß
Obtained from MLAB fit of data to equation 5 (see Methods)
A B
Equation 6 (see Methods) Equation 7 (see Methods)
t~(a) 3 t~s(ß) 3 v~4 x
0 .693/a O .693/ß o /(A + B)
Calculated Value 16 .2 ng/ml 6 .28 ng/ml-1 .063 min . -1 .016 min . 366 ng/ml 35 .6 ng/ml 11 .0 min . 44 .2 min . .256 1/kg
1 A11 parameters obtained from poet-infusion phase, after 14 .2 ug/kg/hr d2HVA for six hours . Body weight was 5 .0 kg . 2 A11 equations obtained from Gibaldi and Perrier (20) 3 Half-life of initial and terminal phases, respectively `'Volume of distribution of the central compartment S Apparent volume of distribution
2346
HVA in Monkey Plasma, Urine and CSF
Pharmacokinetic Parametern of d2-HVA :
Vol . 23, No . 23, 1978
The time course of the decline
of d2-HVA in the plasma after cesnatioa of infusion ie shown in Figure 3. Since a semi-logarithmic plot of d2-HVA concentration in plasma versus time is clearly biexponential, a two-compartment model was assumed to dencribe these data . Table 2 shows the disposition parameters calculated from these data . The half-life of the terminal phase [tß(ß)] ie 44 .2 minutes, which is in good agreement with that found in man after a bolus injection of d2-HVA by Anggard et al . (27) . Even though these investigators ignored the initial rapid phase of HVA distribution, the apparent volume of distribution (VB) of d2-HVA is this monkey of 0 .813 1/kg is reasonably close to the mean of 0.59 1/kg in man (27) . The distribution of d -HVA in both man and monkey after intravenous bolus injection is probably beet 2described by a three-exponential function, if the initial distribution phase is seen with rapid early sampling (unpublished observations) . Experiments are currently in progress to assess the relative importance of each compartment to the total body turnover . CONCLUSIONS HVA in the urine is derived from HVA in the blood and not directly from renal metabolism of DA . Less than one percent of HVA in either lumbar or ventricular CSF appears to be derived from the blood . The disposition of plasma HVA in the monkey fits at least a two-compartment model, assuming that the kinetics of HVA are not altered with enlargement of the body pool of HVA by infusion of d2-HVA . ACKNOWLEDGEMENTS We thank Mr . Lyle Modlin for expert technical assistance and Mr . Walter Chmelewski and Mn . Melody Herbat for excellent assistance with the chemical assays . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
T . NAGATSU, Biochemistry of Catecholamines , p . 260, University Park Press, Baltimore (1973) . L . HELSON, M. FLEISHER, V . BETRUNE, M .L . MURPHY, and M.K . SCHWARTZ, Clin . Chem . _18 613-615 (1972) . S . IMASHUKU, H . TARADA, T . SAWADA, T . NAKAMURA, and E .H . LaBROSSE, Cancer _36 450-457 (1975) . M.A . BREWSTER, D .H . BERRY, and M. MORIARTY, Clin . Chew . _23 2247-2249 (1977) . W . JANUSZEWICZ, M . SZNAJDERMAN, B . WOCIAL, J . PREIBISZ, and W. POPLAWSKA, Clin . Chim . Acts . _32 261-264 (1971) . J .L . CUCHE, 0 . KUCHEL, A. BARBEAU, and J . GENEST, Can. Med . Aseoc. J . _22 443-446 (1975) . U .K . RINNE, V . SONNINEN, and J . PALO, Paychiat, et Neurol . (Basel) _151 321-327 (1966) . A. BARBEAU, Agressologie _9 195-200 (1968) . J .A .R . LENMAN, M .J . TURNBULL, A . REID, and A.M . FLEMING, J, Neurol . Sci . _32 219-225 (1977) . U.S . VON EDLER, I . FLOODING, and F . ISCHAJRO, Ac ta . Soc. Med . U~nal . _64 217-225 (1959) . R .W . ALEXANDER, J .R . GILL, Jr ., H. YAMABE, W. LOVENBERG, and H.R, KEISER, J . Clin . Invest . _54 194-200 (1974) . 0 . KUCHEL, J .L . CUCHE, N .T . BW, and J. GENEST, Am . J. Med . Sci . _272 263-268 (1976) . B . FAUCHEUX, N .T . BUD, and 0 . KUCAEL, Am . J. Phyaiol . 232 F123-F127 (1977) .
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14 . 15 . 16 . 17 . 18 . 19 . 20 . 21, 22, 23 . 24 . 25 . 26, 27 .
in Monkey Plasma, Urine and CSF
2347
L .I . GOLDBERG, Pharmacol, Rev . 24 1-24 (1972) . M . SANDLER and C .R .J . RUTHVEN, Px~ress in Medicinal Chemistry , vol . b, pp , 200-265, Plen~ Press, New York (1959} . G . BARTHOLINI, A . PLETSCH$R, and R . TISSOT, Ex_perientia 22 609-610 (1966) . E . GORDON, M . PERId)W, J . OLIVER, M . EBERT, and I . KOPIN, Neurochem. 2S 347-349 f197S} . D .J . JENDEN, M, ROCH, and R,A, BOOTH, Anal . Biochem, SS 438-448 (1973} . G .D . KNOTT and D .R, REECE, Proceedin a of the ONLINE X 72 International Conference , vol, 1, pg . 497-526, Brunel University, England 1972 . M . GIBALDI and D . PERRIER, Pharmacokinetics , pp . 45-80, Marcel Dekker, Inc ., New York {1975) . B . LINDSTROM, B, S30QUIST, and E . ANGGARD, J . Labelled Comp . 10 187-194 (1974) . M .L . AIEENSTEIN, and J . KORF, Brain Res , 1 49 129-140 (1978) . M. BULAT, M . JAKÜPCEVZC, and 2 . LACKOVIC,Neurosciences Abstracts 4 156 (1974} . H .C . GOLDBERG, and C,M . YATES, Brat . J . Pharmscol . 33 457-471 (196$} . A. PLETSCHER, G, ~ARTHOLINI, and R . TISSOT, Brain Res , _4 106-109, (1967} . L, PROCKOP, S . FAHN, and P . BARHOUR, Brais Res , 80 435-442 (1974} . E . ANGGARD, T, LEWANDER, and B . SJOQUZST, Life Sci . 1$ 111-122 (1974) .
J.
