EFFECT OF HUNTINGTON'S AND ALZHEIMER'S DISEASES ON THE TRANSPORT OF NICOTINIC ACID OR NICOTINAMIDE ACROSS THE HUMAN BLOOD-BRAIN BARRIER L. V. Hankes, H.H. Coenen, E. Rota, K.J. Langen, H. Herzog, W. Wutz, G. Stoecklin, and L.E. Feinendegen Institut fur Medizin Institut fur Chemie I Kernforschungsanlage Julich GmbH D-5170 Julich Germany INTRODUCTION Although Elvehjem et al. (1937) discovered that nicotinamide relieved the neurological symptoms of pellagra in dogs, and in spite of the discovery by Hankes and Elvehjem (1949) that nicotinamide or tryptophan reversed the neurological syndrome produced in rats by a diet high in phenylalanine, the importance of nicotinamide and related compounds in neurology remained dormant for almost two decades. The recent surge of interest in the effects of the tryptophan-kynurenine-nicotinamide pathway compounds in neurology (Stone and Connick, 1985) and the transport of these compounds through the bloodbrain-barrier prompted a study of the transport of nicotinic acid or nicotinamide through the blood-brain barrier and a subsequent study of the rate of metabolism of these two compounds in the human brain in various neurological disease states. COMPOUNDS AND SUBJECTS Carboxy-llC-nicotinic acid used in the study was synthesized by a carboxylation of 3Li-pyridine with llC0 2 produced by a minicyclotron. Carboxamide-llC-nicotinamide was made by amidation of carboxyl-llC-nicotinic acid. Both compounds were purified with a high-pressure liquid chromatography system and dissolved in pyrogen-free saline solution (Machulla et al., 1976). Table 1 shows that 1.8 to 15 mCi of a labeled compound was intravenously injected into normal volunteers and patients with Huntington's or Alzheimer's disease. EXPERIMENTAL METHODS The heads of the subjects were centered in a Scandtronix-type PC 4096-15 WB high resolution (5 mm) positron emission tomographic camera system containing eight rings. This orientation provided 15 sections of an organ at anyone time. Positron emission tomography (PET) images were produced over a period of 40 to 60 minutes after the injection of a labeled compound. These images were used to construct time-activity curves of the activity concentraKynurenine and Serotonin Pathways Edited by R. Schwarcz el al., Plenum Press, New York, 1991
675
Subj ects, compounds and dosages (mCi)
Table 1.
Code
Carboxyl-llCnicotinic acid
Control
lA lB
9.0
Huntington
2A 2B
15.0
Alzheimer
3A
8.0
Carboxamide _llC_ nicotinamide 3.4 5.3 2.5 1.8
tion in the cerebral tissue. Blood samples were drawn at specified intervals for the measurement of the activity present in whole blood and plasma. Pictures of the scans were printed out about 15 minutes after the administration of the compound (Rota et al., 1988). RESULTS AND DISCUSSION The K3 values in Table 2 (rate constants of flow of substrate into metabolism) show that nicotinic acid and nicotinamide passed through the bloodbrain barrier and were slowly accumulated in cerebral tissue. As seen in Table 3, the cerebral uptake of nicotinamide was much higher than that for nicotinic acid. The values in Table 4 show that when the activity in the cerebrum was compared to the activity in plasma, the differences in uptake became smaller than when the uptake was compared to the activity in the injected dose. Apparently, more of the nicotinic acid entered other tissues such as red blood cells. This is again illustrated by the data in Table 5, where the ratio of the activities in blood vs. plasma steadily increased when nicotinic acid was given to the subjects. The values of the ratio for the samples from patients receiving nicotinamide were much lower than those for the samples from patients given nicotinic acid. The data showed that after a slight drop at Table 2.
Rate constants K3 determined by Patlak plot (l/min) Code
Carboxyl-llCnicotinic acid
Control
lA lB
0.040
Huntington
2A 2B
0.012
Alzheimer
3A
0.020
Carboxamide _llC_ nicotinamide 0.024 0.022 0.022 0.012
K3: Rate constant of flow of substrate from compartment of free tracer in tissue to compartment of metabolized tracer in tissues. Patlak plot: Graphic evaluation of transfer to blood flow (Patlak and Blasberg, 1985).
676
Table 3.
Cortical activity concentration per injected dose Code
Carboxyl-llCnicotinic acid
Control
lA lB
3.6
Huntington
2A 2B
2.7
Alzheimer
3A
3.8
(~Ci/ml/mCi)
Carboxamide _llC_ nicotinamide
17.7 17.9 16.8 9.4
three minutes post injection, the values returned to a constant level, suggesting some sort of equilibrium status for this period of time. The large difference in the ratio for nicotinic acid compared to the ratio for nicotinamide again suggests that more of the nicotinamide went into the red cells. There were no significant differences in the observations on the human control subjects compared to the Huntington or Alzheimer patients. SUMMARY
The cerebral uptake of llC-nicotinic acid elC-NAC) and llC-nicotinamide (llC_NAM) was quantified by the use of PET. Based on the amount of activity injected, the PET images showed a low cerebral uptake of llC-NAC, while llC_ NAM was clearly visualized in the cortical areas. This discrepancy was found to be the result of the binding of llC-NAC to the red blood cells by a factor of 5 to 20 above that for llC_NAM. llC_NAM was better extracted by the cerebrum than llC-NAC, as shown by the mean values of the cortical tissue/plasma ratio of 1.9 for llC_NAC and 5 for llC_NAM at 30 min. post-injection. An analysis of Patlak-Gjedde plot curves revealed a metabolic compartment for llC-NAC and llC_NAM with similar values of about 0.02 l/min for the accumulation constant K3. This was indicative of a slower transport rate for llC_ NAC. A significant finding of the study was the increasing ratio of activity concentrations in red blood cells versus the concentrations in plasma (over time). There were no significant differences between the data from normal volunteers and patients with Huntington's or Alzheimer's disease.
Table 4.
Ratio of cortical activity concentration/plasma activity concentration (mean between 20-40 min., p.i.) (~Ci/~Ci) Code
Control Huntington Alzheimer
Carboxy1- llC_ nicotinic acid
lA lB
2.0
2A 2B
2.0
3A
1.7
Carboxamide _llC_ nicotinamide
3.5 4.5
8.0
4.0
677
Table 5. Sample Time (min) 0.25 0.5 0.75 1.0 1.5 2.0 3.0 4.0 6.0 10.0 15.0 20.0 25.0 30.0 40.0 45.0 50.0 60.0
Ratio of whole blood vs. plasma activity (nCi/ml) Nicotinic acid 2A
3A 2.5
14.2
4.9 5.5 10.8
25.9
13.7 16.7
40.3 47.1 59.9
20.8
Nicotinamide 2B
3A
IB
lA
0.74 0.70 2.26 1.63 1.25 1.18 1.28 1.64 2.91 3.04 3.31 3.34 3.33 3.74
1.02 0.80 2.47 2.29 2.11 1. 95 2.39 3.31 2.85 3.64 3.74 3.32 3.61
1.24 1.18 1.10 1.14 1.07 1.02 1.05 1.13 1.20 1.45 1.64 1. 79 1.91 1. 99
0.73 0.62 0.93 1.20 1.09 1.01 0.98 1.00 1.10 1.18 1.27 1.47 1.46 1. 35
3.99
3,76
2.18
1. 52
3.31
3.25
2.12
1.53
The number-letter code is the same as in Table 1. REFERENCES E1vehjem, C.A., Madden, R.J., Strong, F.M., and Wollen, D.W., 1937, Relation of nicotinic acid and nicotinic acid amide to canine black tongue, J. Am. Chem. ~oc., 59:1767-1768. Hankes, L.V., and E1vehjem, C.A., 1949, A nervous syndrome produced with phenylalanine and methionine, Proc. Soc. Exp. BioI. Hed., 72:349-351. Machu11a, H.J., Laufer, P., and Stoeck1in, G., 1976, Radioanalytica1 quality control of llC, 1BF and 123 1 labelled compounds and radiopharmaceutica1s, Radioana1. Chem., 32:32:381-400. Pat1ak, C.S., and B1asberg, R.G., 1985, Graphical evaluation of blood to brain transfer constants from multiple-time uptake data, J. Cereb. Blood Flow Hetab., 5:584-590. Rota, E., Herzog, H., Schmid, A., Holte, S., and Feinendegen, L.E., 1988, Performance of machine parameters and its influence on metabolic data using the PET-scanner Pc-4096-15WB, J. Nuc1. Hed., 29:877. Stone, T.W., and Connick, J.H., 1985, Quinolinic acid and other kynurenines in the central nervous system, Neuroscience, 15:597-617.
678
675
Subj ects, compounds and dosages (mCi)
Table 1.
Code
Carboxyl-llCnicotinic acid
Control
lA lB
9.0
Huntington
2A 2B
15.0
Alzheimer
3A
8.0
Carboxamide _llC_ nicotinamide 3.4 5.3 2.5 1.8
tion in the cerebral tissue. Blood samples were drawn at specified intervals for the measurement of the activity present in whole blood and plasma. Pictures of the scans were printed out about 15 minutes after the administration of the compound (Rota et al., 1988). RESULTS AND DISCUSSION The K3 values in Table 2 (rate constants of flow of substrate into metabolism) show that nicotinic acid and nicotinamide passed through the bloodbrain barrier and were slowly accumulated in cerebral tissue. As seen in Table 3, the cerebral uptake of nicotinamide was much higher than that for nicotinic acid. The values in Table 4 show that when the activity in the cerebrum was compared to the activity in plasma, the differences in uptake became smaller than when the uptake was compared to the activity in the injected dose. Apparently, more of the nicotinic acid entered other tissues such as red blood cells. This is again illustrated by the data in Table 5, where the ratio of the activities in blood vs. plasma steadily increased when nicotinic acid was given to the subjects. The values of the ratio for the samples from patients receiving nicotinamide were much lower than those for the samples from patients given nicotinic acid. The data showed that after a slight drop at Table 2.
Rate constants K3 determined by Patlak plot (l/min) Code
Carboxyl-llCnicotinic acid
Control
lA lB
0.040
Huntington
2A 2B
0.012
Alzheimer
3A
0.020
Carboxamide _llC_ nicotinamide 0.024 0.022 0.022 0.012
K3: Rate constant of flow of substrate from compartment of free tracer in tissue to compartment of metabolized tracer in tissues. Patlak plot: Graphic evaluation of transfer to blood flow (Patlak and Blasberg, 1985).
676
Table 3.
Cortical activity concentration per injected dose Code
Carboxyl-llCnicotinic acid
Control
lA lB
3.6
Huntington
2A 2B
2.7
Alzheimer
3A
3.8
(~Ci/ml/mCi)
Carboxamide _llC_ nicotinamide
17.7 17.9 16.8 9.4
three minutes post injection, the values returned to a constant level, suggesting some sort of equilibrium status for this period of time. The large difference in the ratio for nicotinic acid compared to the ratio for nicotinamide again suggests that more of the nicotinamide went into the red cells. There were no significant differences in the observations on the human control subjects compared to the Huntington or Alzheimer patients. SUMMARY
The cerebral uptake of llC-nicotinic acid elC-NAC) and llC-nicotinamide (llC_NAM) was quantified by the use of PET. Based on the amount of activity injected, the PET images showed a low cerebral uptake of llC-NAC, while llC_ NAM was clearly visualized in the cortical areas. This discrepancy was found to be the result of the binding of llC-NAC to the red blood cells by a factor of 5 to 20 above that for llC_NAM. llC_NAM was better extracted by the cerebrum than llC-NAC, as shown by the mean values of the cortical tissue/plasma ratio of 1.9 for llC_NAC and 5 for llC_NAM at 30 min. post-injection. An analysis of Patlak-Gjedde plot curves revealed a metabolic compartment for llC-NAC and llC_NAM with similar values of about 0.02 l/min for the accumulation constant K3. This was indicative of a slower transport rate for llC_ NAC. A significant finding of the study was the increasing ratio of activity concentrations in red blood cells versus the concentrations in plasma (over time). There were no significant differences between the data from normal volunteers and patients with Huntington's or Alzheimer's disease.
Table 4.
Ratio of cortical activity concentration/plasma activity concentration (mean between 20-40 min., p.i.) (~Ci/~Ci) Code
Control Huntington Alzheimer
Carboxy1- llC_ nicotinic acid
lA lB
2.0
2A 2B
2.0
3A
1.7
Carboxamide _llC_ nicotinamide
3.5 4.5
8.0
4.0
677
Table 5. Sample Time (min) 0.25 0.5 0.75 1.0 1.5 2.0 3.0 4.0 6.0 10.0 15.0 20.0 25.0 30.0 40.0 45.0 50.0 60.0
Ratio of whole blood vs. plasma activity (nCi/ml) Nicotinic acid 2A
3A 2.5
14.2
4.9 5.5 10.8
25.9
13.7 16.7
40.3 47.1 59.9
20.8
Nicotinamide 2B
3A
IB
lA
0.74 0.70 2.26 1.63 1.25 1.18 1.28 1.64 2.91 3.04 3.31 3.34 3.33 3.74
1.02 0.80 2.47 2.29 2.11 1. 95 2.39 3.31 2.85 3.64 3.74 3.32 3.61
1.24 1.18 1.10 1.14 1.07 1.02 1.05 1.13 1.20 1.45 1.64 1. 79 1.91 1. 99
0.73 0.62 0.93 1.20 1.09 1.01 0.98 1.00 1.10 1.18 1.27 1.47 1.46 1. 35
3.99
3,76
2.18
1. 52
3.31
3.25
2.12
1.53
The number-letter code is the same as in Table 1. REFERENCES E1vehjem, C.A., Madden, R.J., Strong, F.M., and Wollen, D.W., 1937, Relation of nicotinic acid and nicotinic acid amide to canine black tongue, J. Am. Chem. ~oc., 59:1767-1768. Hankes, L.V., and E1vehjem, C.A., 1949, A nervous syndrome produced with phenylalanine and methionine, Proc. Soc. Exp. BioI. Hed., 72:349-351. Machu11a, H.J., Laufer, P., and Stoeck1in, G., 1976, Radioanalytica1 quality control of llC, 1BF and 123 1 labelled compounds and radiopharmaceutica1s, Radioana1. Chem., 32:32:381-400. Pat1ak, C.S., and B1asberg, R.G., 1985, Graphical evaluation of blood to brain transfer constants from multiple-time uptake data, J. Cereb. Blood Flow Hetab., 5:584-590. Rota, E., Herzog, H., Schmid, A., Holte, S., and Feinendegen, L.E., 1988, Performance of machine parameters and its influence on metabolic data using the PET-scanner Pc-4096-15WB, J. Nuc1. Hed., 29:877. Stone, T.W., and Connick, J.H., 1985, Quinolinic acid and other kynurenines in the central nervous system, Neuroscience, 15:597-617.
678
