Carnitine, Carnitine Acetyltransferase, and Glutathione in. Alzheimer Brain. Tapas K. Makar, 1 Arthur J. L. Cooper, ~,2,s Beth Tofel-Grehl, 3 Howard T. Thaler, 4.
Neurochemical Research, Vol. 20, No. 6, 1995, pp. 705-711
Carnitine, Carnitine Acetyltransferase, and Glutathione in Alzheimer Brain Tapas K. Makar, 1 Arthur J. L. Cooper, ~,2,s Beth Tofel-Grehl, 3 H o w a r d T. Thaler, 4 and John P. Blass 2,3
(Accepted January 30, 1995)
Glutathione and "total" carnitine (i.e., free camitine plus acid-soluble camitine esters) were measured in an affected (superior frontal gyrus; SFG) and unaffected (cerebellum: CBL) region of Alzheimer disease (AD) and control brains. Average glutathione content in AD SFG (n = 13) and AD CBL (n = 7) (7.9 _+ 2.1 and 11.9 _ 4.0 nmol/mg protein, respectively (mean ___ S.D.)) was similar to that in control SFG (n = 13) and CBL (n = 6) (7.7 _+ 2.0 and 11.6 +_ 2.6 nmol/mg protein, respectively). However, glutathione increased significantly with age in AD brain (p = 0.003) but not in control brain. Average total camitine in AD SFG (84 _+ 47 pmol/mg protein; n = 10) and AD CBL (108 ___ 86 pmol/mg protein; n = 7) was not significantly different from that in the corresponding regions of control brain (148 _ 97 (n = 10) and 144 _ 107 (n = 6) pmol/ mg protein, respectively). However, a significant decline of total carnitine with age in both regions was noted for AD brain, but not for control brain. Carnitine acetyltransferase activity in the AD SFG (n = 13) was not significantly different from that of control SFG (n = 13) (1.83 _+ 1.05 and 2.04 _+ 0.82 nmol/min/mg protein, respectively). However, carnitine acetyltransferase activity of AD CBL (n = 7) was significantly lower than that of control CBL (n = 6) (1.33 _+ 0.88 versus 2.26 + 0.66 nmol/mirgmg protein; p = 0.05).
KEY WORDS: Alzheimer's disease; camitine; camitine acetyltransferase; glutathione, brain.
camitine acetyltransferase (CarAT) activity in AD cerebral microvessels and in three of four regions of AD brain is significantly reduced compared with non-AD brain (9). However, Perry et al. previously found no differences in carnitine acetyltransferase activity in an affected region and in an unaffected region in Alzheimer disease (AD) brain (10). CarAT catalyzes the reversible formation o f the acyl ester o f camitine from acetyl-Coenzyme A (acetyl-CoA) and other short-chain fatty acyl CoAs. The enzyme is located both in mitochondria and in peroxisomes (7). The enzyme is involved in the transport o f active acetyl groups from the inner mitochondrial compartment into the cytoplasm. Disruption o f glutathione and carnitine metabolism in AD brain is expected to result in an exacerbation o f mitochondrial abnormalities. Therefore, it was consid-
INTRODUCTION Replicated studies have demonstrated several biochemical abnormalities in AD brain mitochondria (1-6). Camitine is involved in the transport o f long-chain fatty acyl moieties into the mitochondria for [3 oxidation (7). Glutathione is important for mitochondrial function and integrity (8). Recently, Kalaria and Harik reported that Departments of 1Biochemistry and 2Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021. 3 Burke Medical Research Institute, White Plains, NY 10605. 4 Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021. 5 To whom to address reprint requests: Dr. Arthur J. L. Cooper, Department of Biochemistry, Comell University Medical College, 1300 York Avenue, New York, NY 10021.
705 0364-3190/95/0600-0705507.50/0 9 1995PlenumPublishingCorporation
706
Makar, Cooper, Tofel-Grehl, Thaler, and Blass
Table L Individual Values of Age at Death, Sex, Postmortem Interval (PMI), Carnitine Acetyltransferase (CarAT), Glutathione (GSH), and Carnitine (Car) for all Subjects in the Study SFG
AD M F M F F F M M M M M F M CON Mh Fh Fs Mh Ms Fa Fh M' Fh Mh M" M" F~
Age
PMI
CarAT
GSH
78 93 77 76 79 76 73 78 92 72 81 85 82
2.0 2.6 2.3 1.5 3.5 3.0 3.25 2.0 2.5 2.5 1.75 3.0 2.5
2.61 0.98 0.30 1.48 1.17 1.99 1.97 4.20 2.92 0.59 1.87 1.39 2.42
5.93 6.46 10.7 8.23 6.33 9.55 5.65 7.82 10.3 3.99 9.54 8.22 9.91
80 94 95 79 77 73 85 77 76 77 75 85 76
1.6 2.5 3.75 2.3 2.0 2.25 1.00 2.0 1.25 2.0 1.5 2.5 1.5
2.86 2.67 2.79 1.26 1.21 1.13 1.03 3.00 2.78 1.26 1.61 1.85 3.05
6.33 6.44 6.53 6.97 5.22 7.22 8.00 7.57 4.95 9.81 10.9 11.0 8.83
Cerebellum Car
90 94 140 118 49 46 174 44 54 34
298 178 111 69 291 73 25 178 56 199
CarAT
GSH
Cam
1.08 0.58 0.30
7.75 16.5 11.6
142 17 80
2.10 1.21 2.85 1.20
7.67 7.67 11.4 11.7
284 284 87 87
2.27 1.39 3.13 2.18
8.14
348
12.3 9.54
77 122
13.9 11.0 14.8
159 111 49
1.71 2.86
AD, Alzheimer disease; CON, controls. The causes of death among the controls is as follows: aadenocarcinoma, hheart disease, "renal disease and 'stroke. Units are defined in Tables II-IV. Missing values are due to insufficient tissue for complete analyses or to unavailability of cerebellar tissue from some AD and controls.
e r e d i m p o r t a n t to m e a s u r e t h e l e v e l s o f t h e s e t w o s m a l l molecular weight compounds and the activity of CarAT in A D b r a i n . D e t a i l s o f t h e s e s t u d i e s are p r e s e n t e d b e low.
of ice-cold, 50 mM Tris-HC1 buffer (pH 7.4). The homogenate was centrifuged at 3,000 g (20 mins, 4~ and the precipitate was discarded. After further centrifugation (12,000 g, 20 mins, 4~ the pellet was resuspended in ice-cold 50 mM Tris-HC1 buffer and assayed at 25~ by the spectrophotometrie procedure outlined by Bergmeyer et al. (12) using double-beam speetrophotometry (Cary model 219). The balance (control) cuvette routinely contained all components of the reaction mixture except carnitine. Control experiments indicated that measured CarAT activity was identical whether the routine (no carnitine) control or a no-enzyme control was used. For measurement of carnitine, frozen brain samples were homogenized in 10 vols of ice-cold 5% (w/v) 5-sulfosalicylic acid (SSA). After centrifugation (12,000 g), 1 M NaOH was added to the supernatant to yield a final pH of 9.0. The solution was allowed to stand for 5 hours at room temperature to ensure complete hy&'olysis of carnitine esters; carnitine is stable under these conditions. The hydrolysate was analyzed for "total" acid-soluble carnitine (i.e., free camitine plus small- to medium-length acyl camitines) by the method of Bieber mad Lewin (13). The procedure used precipitates long-chain carnitine esters, but the contribution of long-chain carnitine esters to the total is small (7,13). In mammals, carnitine is not metabolized except by intestinal flora (7). Therefore, it is likely that total carnitine concentration is essentially stable in post-mortem brain. However, the degree of acylation of carnitine is likely to vary with post-mortem interval. Therefore, only total acid-soluble camitine was measured in the present experiments. Total glutathione (i.e., glutathione (GSH) plus glutathione disulfide (GSSG, oxidized glutathione)) was measured in the SSA supematant by the method of Tietz (14) as modified by Cooper et al (15). The GSH/GSSG ratio was not measured in the present experiments because of the likelihood of post mortem artifact. Under normal conditions
