| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Technical Advance |
From the Pathophysiology Department,* Institute forNeuroscience, Tongji Medical School, Huazhong University of Science andTechnology, Wuhan, People’s Republic of China; and the Department ofNeurochemistry, New York State Institute forBasic Research in Developmental Disabilities, Staten Island, New York
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
400 and 1300 times more sensitive than
conventional enzyme-linked immunosorbent assay in determining the
hyperphosphorylated tau and total tau, respectively. With this
method, we measured both total tau and tau phosphorylated at
Ser-396/Ser-404 in lumbar CSFs from AD and control patients. We found
that the total tau was 215 ± 77 pg/ml in cognitively normal
control (n = 56), 234 ± 92 pg/ml in
non-AD neurological (n = 37), 304 ±
126 pg/ml in vascular dementia (n = 46),
and 486 ± 168 pg/ml (n = 52) in AD
patients, respectively. However, a remarkably elevated
level in phosphorylated tau was only found in AD (187 ± 84
pg/ml), as compared with normal controls (54 ± 33
pg/ml), non-AD (63 ± 34 pg/ml), and vascular
dementia (72 ± 33 pg/ml) groups. If we used the ratio of
hyperphosphorylated tau to total tau of 
0.33 as cutoff for AD
diagnosis, we could confirm the diagnosis in 96% of the
clinically diagnosed patients with a specificity of 95%,
86%, 100%, and 94% against nonneurological,
non-AD neurological, vascular dementia, and all of the
three control groups combined, respectively. It is suggested
that the CSF level of tau phosphorylated at Ser-396/Ser-404 is a
promising diagnostic marker of AD.
Among all of the abnormalities described in the AD brain to date, those related to the hallmark neuropathological lesions, ie, formation of neurofibrillary tangles and deposition of amyloid ß, are the best documented and the most promising diagnostic markers. In addition to a decreased level of Aß1-42,7 a pronounced increase in CSF tau has been found in most AD patients.5,6,8-13 However, an increased level of total tau is also found in several neurological disorders other than AD.
It has been well studied and commonly accepted that abnormally phosphorylated tau is the major protein subunit of Alzheimer’s paired helical filaments (PHFs).14,15 Among all of the phosphorylation sites found in PHF-tau,16 C-terminal Ser-396 and Ser-404 represent a major Alzheimer’s epitope. Phosphorylation of tau at this epitope reduces its biological activity in promoting microtubule assembly, binding to microtubules, and the ability in stabilizing microtubules against nocodazole-induced depolymerization.17-19 Dephosphorylation of AD abnormally hyperphosphorylated tau (AD P-tau) at these sites by protein phosphatases shifts its mobility to the position of normal tau in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, restores its biological activity, and relaxes the structure of PHFs.20,21 All these data strongly suggest that phosphorylation at Ser-396 and Ser-404 of tau might play a crucial role in AD pathology. However, the level of phosphorylated tau in CSF is relatively low compared with normal tau and has been difficult to quantitate.22 To this end, we have modified and adapted the enzyme amplification method of Johannsson and colleagues,23 and successfully developed a highly specific and ultrasensitive assay in the attomolar range for the quantitation of total tau and tau phosphorylated at Ser-396/Ser-404 in CSF, and have found a significant increase in the levels of tau, especially the phosphorylated protein in AD.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Samples of lumbar CSF of living patients were obtained from The
Netherlands Brain Bank and several teaching hospitals in China (THC)
(Table 1)
. The information on CSF samples
obtained from The Netherlands Brain Bank is as follows: AD
(n = 30), 13 male and 17 female with ages from
62 to 78 years (mean, 71 years), Mini-Mental State Examination (MMSE)
score from 8 to 27 (mean, 21.4), and ApoE genotypes 4/4
(n = 4), 4/3 (n = 14),
3/3 (n = 11), and 3/2
(n = 1); vascular dementia (VaD,
n = 18); non-AD (n = 13): spinal
canal stenosis (n = 4), depression
(n = 3), malignant lymphoma
(n = 2), vertebro-basilar artery infarct
(n = 1), diabetic neuropathy
(n = 1), subclavian steal syndrome
(n = 1) and polyneuritis and ataxia
(n = 1). The samples collected in China were
from the Tongji Medical University-affiliated Tongji Hospital: AD
(n = 9), aged control (n
= 14), Tongji Hospital-affiliated Qiaoko Military Hospital: aged
control (n = 16), the second Hospital in Wuhan
City: AD (n = 4), and the 187 Military Hospital
in Haiko City: AD (n = 9), VaD
(n = 28), age-matched non-AD neurological
control (n = 24): meningeal hemangioma
(n = 3), meningitis (n =
2), ataxia (n = 2), spinal canal stenosis
(n = 1), diabetic polyneuropathy
(n = 3), depression (n =
4), multiple sclerosis (n = 1), myodystrophy
(n = 1), Parkinson syndrome
(n = 2), acoustic neurinoma
(n = 1), vertebro-basilar artery infarct
(n = 2) and malignant lymphoma
(n = 2). The diagnosis was made by different
mental performance tests designed by different hospitals, computed
tomography or magnetic resonance imaging, disease history, family
history, clinical signs, and symptoms.
|
Recombinant human brain tau410 (tau 39 clone) and from an AD brain the AD P-tau were purified as described previously.24,25 Resazurin, resorufin, NAD+-free nicotinamide adenine dinucleotide phosphate (NADP+), alkaline phosphatase (EC 3.1.3.1), NADH-dependent diaphorase (EC 1.8.1.4), and NAD+-dependent alcohol dehydrogenase (EC 1.1.1.1) were all purchased from Sigma (St. Louis, MO). The capture polyclonal antibody 92e was described previously.26 Monoclonal antibodies Tau-1 (to tau unphosphorylated at Ser-198/199/202) and PHF-1 (to tau phosphorylated at Ser-396/404) were generous gifts from Drs. L. Binder (North Western University, Chicago, IL) and P. Davies (Albert Einstein College of Medicine, Bronx, NY), respectively. The alkaline phosphatase-conjugated goat anti-mouse IgG used as secondary antibody was purchased from Jackson (West Grove, PA). The major equipment used was Hitachi model 850 fluorescence spectrophotometer (Chiyoda-ku, Tokyo, Japan) and Shimadzu UV-120-02 spectrophotometer (Kyoto, Japan).
Procedure for Bienzyme-Substrate-Recycle Enzyme-Linked Immunosorbent Assay (ELISA)
Step 1: Coating with Capture Antibody
Coat the microtiter plate with tau antiserum 92e (1:2500), 100 µl/well for overnight at 4°C. Wash the plate five times with Tris-buffered saline (TBS), pH 8.5, containing 0.05% Tween 20 (TTBS).
Step 2: Blocking
Add blocking solution (TTBS plus 3% bovine serum albumin), 150 µl/well, and incubate at 37°C for 1 hour, wash as above.
Step 3: Addition of Antigen
Add 20 µl of CSF, diluted to 100 µl in TTBS, 3% bovine serum albumin, and 0.02% NaN3 to the plate, incubate overnight at 4°C, and wash as above.
Step 4: Addition of Reporter/Primary Antibody
Add 100 µl of Tau-1 (1:50,000) or PHF-1 (1:200) to the plate, incubate at 37°C for 1 hour, and wash as above.
Step 5: Addition of Secondary Antibody
Add 100 µl of alkaline phosphatase-conjugated goat anti-mouse IgG (1:10,000) to the plate, incubate at 37°C for 1 hour, and wash as above.
Step 6: Initiating Reaction
Add 100 µl of freshly prepared initiation buffer (225 mmol/L diethanolamine, pH 9.5, 0.04 mmol/L MgCl2, and 1 µmol/L NADP+) to the plate and incubate at 30°C for 45 minutes.
Step 7: Bienzyme Substrate Recycle
Transfer the initiation mixture to an Eppendorf tube and add 100 µl of bienzyme substrate recycling solution [0.1 mol/L phosphate buffer, pH 7.4, 6% alcohol, 0.5 U/ml alcohol dehydrogenase, 0.0125 U/ml diaphorase, and 8 µmol/L resazurin (the enzymes and the substrate prepared just before use)]. Incubate at 37°C for 30 minutes.
Step 8: Stopping the Reaction and Measuring the Fluorescence
The reaction is stopped by boiling the reaction mixture for 5 minutes. The fluorescence is then measured at excitation at 560 nm and emission at 590 nm. For conventional ELISA, 4-methylumbelliferyl phosphate (4-Mu-P) was used as the substrate of alkaline phosphatase-conjugated secondary antibody and fluorescence produced by 4-Mu was measured at excitation of 385 nmol/L and emission of 448 nmol/L.
For determination of total tau levels by antibody Tau-1, each CSF sample, before use in the above assay, was dephosphorylated by alkaline phosphatase (2000 U/ml) for 6 hours at 37°C in buffer, containing 50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L MgCl2, 1% ß-mercaptoethanol, and 0.5 mmol/L phenylmethyl sulfonyl fluoride. Recombinant human brain tau410, and ADP-tau purified from an AD brain were used as standards for total tau and phosphorylated (at Ser-396/404) tau, respectively.
Statistical Analysis
Assuming taus used for standard curves as 100% pure, and in the case of AD P-tau, 100% phosphorylation at Ser-396/404, the tau levels were calculated as means ± SD. The variation analysis and significance tests among the groups were made by clinical epidemiology statistic software STATE 5.0 (Harvard University, Boston, MA).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The ELISA for total tau and for tau phosphorylated at the PHF-1
site were made ultrasensitive by using bienzyme-substrate recycling.
The principle of this assay is shown in Figure 1
. In this assay
NADP+ used as a substrate is dephosphorylated by
the alkaline phosphatase-conjugated secondary antibody into
NAD+. The NAD+ then accepts
hydrogen from alcohol and is reduced into NADH, and subsequently, NADH
transfers its hydrogen to the substrate resazurin and forms the
fluorescent product resorufin and thereby regenerates
NAD+; this NAD+ then starts
over again the cycle and this process continues in the presence of
excess of resazurin. Bienzymes involved in this cycle are alcohol
dehydrogenase and diaphorase. This last part of the assay is the key
for the high sensitivity of the technique.
|
. By mixing
different concentration of substrate resazurin with resorufin and
measuring the final fluorescence at above wavelength, we found that
resazurin quenched the fluorescence produced by resorufin when the
concentration was greater than 8 µmol/L (Figure 2b)
. To determine the
optimal concentration of the substrates involved in the recycling, we
studied the fluorescent signal generated at different concentrations of
NAD+ at resazurin concentrations of 2 µmol/L, 8
µmol/L, or 64 µmol/L. The most effective concentration match was
found to be 8 µmol/L of resazurin and 100 nmol/L of
NAD+ (Figure 2c)
. At a resazurin concentration of
2 µmol/L, the fluorescent density did not increase even using a high
concentration of NAD+, probably because of a
rapid consumption of resazurin; and at resazurin concentrations greater
than 8 µmol/L, a remarkable quenching and high background were
observed (data not shown). Next we determined the optimal ratio of two
enzymes involved in the recycle. When the ratio of alcohol
dehydrogenase to diaphorase (Dia) was 0.5 U to 0.0125 U, the highest
sensitivity and a widest linear range of NAD+
(0.1 nmol/L to 100 nmol/L) were reached (Figure 2d)
. However, if the
ratio of alcohol dehydrogenase to Dia was set at 0.25 U to 0.006 U or
0.125 U to 0.003 U, the linear range of NAD+ was
shifted to 10 nmol/L to 500 nmol/L and 2 nmol/L to 500 nmol/L,
respectively (not shown), but the sensitivity was markedly reduced
(Figure 2d)
.
|
This reaction provides substrate NAD+
through the hydrolysis of NADP+ that is catalyzed
by the secondary antibody-conjugated alkaline phosphatase. It is a
critical step to link ELISA with the bienzyme-substrate-recycling
reaction. To optimize the conditions for this reaction, we first
checked the factors, which affect the activity of alkaline phosphatase.
It was shown that the activity of alkaline phosphatase was decreased
with an increased concentration of Zn++.
Increased activity was observed by adding Mg++ to
the reaction system. The peak activity was reached when the
concentration of Mg++ and diethylamine was set at
0.04 mmol/L and 225 mmol/L in the absence of Zn++
(data not shown). In addition, the optimum reaction time and
temperature for alkaline-phosphatase activity were determined as 45
minutes at 30°C. When the temperature was increased to 43°C, the
background was high and an inactivation of the enzyme started to occur
at 30 minutes (data not shown). To achieve low background and
reproducible results, it was found extremely important to keep
alkaline-phosphatase substrate NADP+ in an
NAD+-free condition. Therefore, we carefully
determined the condition that would lead to a spontaneous hydrolysis of
NADP+. It was found that a significant hydrolysis
of NADP+ occurred when stored at room temperature
(25 30°C) as solution in water or diethylamine buffer.
Whereas, only a 10 to 15% hydrolysis of NADP+
was observed when its solution was stored at 4°C for 48 hours. The
optimal storage condition was found to be -20°C because even 10
times freeze and thaw cycles did not induce any detectable hydrolysis
of the compound (data not shown). The highest sensitivity and the
widest linear range for alkaline-phosphatase activity were observed at
the NADP+ concentration of 1 µmol/L. At 1
µmol/L of NADP+ concentration, when the
background was subtracted, a fluorescence of 20 and a relative
fluorescence (logarithm value of fluorescence) linear range of 0 to 3
were obtained (data not shown). If the concentration was lower than 1
µmol/L, a high sensitivity but a narrow linear range were seen. On
the other hand, a higher concentration of NADP+
(>1 µmol/L) induced a lower sensitivity and a higher background
(Figure 2e)
.
Optimization of the Immunoreaction in the Bienzyme-Substrate-Recycle ELISA
We optimized the ELISA component of the above assay using 4-methylumbelliferyl phosphate (4-Mu-P) as substrate of alkaline phosphatase and quantitated the fluorescent product 4-Mu. The assay was linear when the concentration of standard 4-Mu was between 0.15 µmol/L to 160 µmol/L (data not shown). The linear range was also observed by using 0.8 mmol/L 4-Mu-P and dilution of alkaline phosphatase-conjugated secondary antibody at 1:1.5 x 107 to 1:2.5 x 105 (data not shown). Additionally, no cross-reaction was found between capture antibody 92e (1:2500 and 1:10,000) and the secondary antibody (1:10,000 and 1:30,000), or Tau-1 (1:10,000 and 1:50,000) or PHF-1 (1:100 and 1:200). Similarly no cross-reaction of Tau-1 to phosphorylated tau purified from AD brain or of PHF-1 to recombinant tau was found (not shown).
Comparison of the Sensitivity of the Bienzyme-Substrate-Recycle ELISA with the Conventional ELISA
After optimizing all above conditions, and by linking the above
three portions of the procedure together we established the
bienzyme-substrate-recycle ELISA. We then compared this assay with the
conventional ELISA using the standard recombinant human tau and AD
P-tau. We found that the sensitivity of the conventional ELISA was 1 ng
tau with a linear range of 1 ng to 32 ng (20 fmol to 640 fmol for
average molecular weight of 50,000) (Figure 3a)
and 0.2 ng AD P-tau with a linear
range of 0.2 ng to 10 ng (4 fmol to 200 fmol) (Figure 3b)
. In contrast,
by the bienzyme-substrate-recycle ELISA, the detection range for normal
tau was 0.75 pg to 200 pg (15 amol to 4 fmol) (Figure 3c)
, which stands
for 1300 times increase in sensitivity and more than five times
enlargement of the detection range compared with conventional ELISA.
For AD P-tau, the detectable range was 0.5 pg to 50 pg (10 amol to 1
fmol) (Figure 3d)
, which equals an 400 times increase in sensitivity
and two times enlargement of the detection range.
|
We measured the levels of total normal tau and of P-tau in lumbar
CSF of patients collected from The Netherlands and from the Peoples
Republic of China by using the assay established in the present study.
It was found that both total tau and P-tau were significantly increased
in AD as compared to cognitively normal, non-AD, and VaD controls
(Figure 4, a and b)
. However, a
statistically significant higher level of total tau was also seen in
VaD than normal controls. In contrast to total tau, a markedly higher
level of P-tau was only seen in AD but not in VaD (Figure 4b)
. The
absolute values for phosphorylated tau in the present study might be
overestimated because AD P-tau used as a standard might not be 100%
phosphorylated at Ser-396/Ser-404. However, the relative values among
different groups should not be affected. No correlation was detected in
AD patients from The Netherlands Brain Bank between the sex (Figure 4c)
, age (Figure 4d)
, MMSE score (Figure 4e)
, and ApoE genotype (Figure 4f)
with the level of tau or P-tau in CSF. Because ApoE genotyping data
were available only on AD patients from The Netherlands Brain Bank only
these cases were used for all correlation analyses. Consistent with
previous studies5,11,12
no significant changes in the
elevation of CSF tau levels were observed between mild (MMSE scores, 26
to 27) and severely impaired (MMSE scores, 8 to 13) AD patients.
|
370 pg/ml (mean + 1.72 SD of normal control value) as
cutoff for AD, 79% sensitivity and specificity of 100%, 95%, and
50% toward normal, non-AD neurological, and VaD samples, respectively,
were achieved (Table 2)
. If we used P-tau
as a diagnostic marker of AD, the sensitivity and especially the
specificity toward VaD group were markedly increased. A P-tau to total
tau ratio of 0.33 as cutoff for AD diagnosis resulted in sensitivity
of 96% and specificity of 95%, 86%, 100%, and 94% toward normal
aged control, non-AD neurological, VaD, and all these three groups
combined, respectively (Figure 5
, Table 2
).
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The level of the hyperphosphorylated tau is too low to be detected by conventional methods. To solve this problem, in the present study we have adapted the enzyme amplification immunoassay of Johansson and colleagues23 that links conventional ELISA with bienzyme substrate recycling and have established a highly specific and ultrasensitive method that can measure AD abnormal tau in CSF. By using this method, we have compared the level of total tau and tau phosphorylated at Ser-396/Ser-404 (PHF-1 site) in AD with VaD, non-AD, and normal-aged control CSFs collected from two different countries. Our findings suggest that the assay developed is sensitive and specific enough for the detection of micro amounts of tau in CSF and that the increased level in CSF of tau phosphorylated at the PHF-1 site is specific to AD and might be used as a diagnostic aid for AD. It may be noted that none of the control groups of patients had any lesions of abnormally hyperphosphorylated tau in their brains and thus consequently low CSF levels of abnormally hyperphosphorylated tau. Cases with non-AD tauopathies such as frontotemporal dementia, corticobasal degeneration, and Pick disease will most likely have elevated CSF levels of abnormally hyperphosphorylated tau.
Tau in AD brain is known to be abnormally hyperphosphorylated at more than 21 sites.16,29 The PHF-1 (Ser-396/404) site investigated in the present study is one of the major and most well-studied sites. Other major abnormally phosphorylated sites include Thr-181, Ser-199, and Ser-231/Thr-235. Similar to our findings the levels of tau phosphorylated at Ser-231/Thr-235,22,30 Ser-199,31 and at Thr-18132 have been found to be specifically elevated in AD CSF. Thus, there is increasing evidence for the promising diagnostic potential of CSF abnormally hyperphosphorylated tau levels in AD.
Conventional ELISA has been widely used to assay micro amounts of antigens and antibodies since it was established in 1966.33 Many efforts have been made to improve the sensitivity of the assay. The main strategies used for this particular purpose have been to amplify the signal produced by antibody-conjugated enzymes, such as to increase the number of copies of the reporter enzyme itself or using better substrates. The sensitivity of the conventional ELISA was increased 2 to 100 times by using the biotin-avidin system because avidin has high affinity to biotin (one avidin binds four biotins), and multiple biotins can be conjugated to the secondary antibodies or enzymes.34 However, nonspecific binding caused by positive charge of the avidin molecule is the major disadvantage of this system. Therefore, neutral streptavidin was introduced to reduce the background.35 In respect to the improvement in sensitivity made with respect to substrates, various approaches, such as the use of fluorescent-, chemiluminescent-, or radioisotope-labeled substrates, have been tested. Fluorescent substrate enlarged the sensitivity of the method by 5 to 100 times,35 and radioimmunoassay made the technique a thousand times more sensitive than conventional ELISA.36 Although radioimmunoassay is reasonably sensitive enough for the determination of micro amounts of proteins, it is limited in use, especially in the clinical application in developing countries, because of the special requirements in reagents, instruments, and proper facilities where radiation can be used. The method developed in the present study increased the sensitivity to 400 or 1300 times toward P-tau or normal recombinant tau, respectively, and it does not need special reagents or equipment. Therefore, it has great potential in clinical application and in drug trials.
According to the principle of the assay shown in Figure 1
, the
following three elements must be carefully addressed to detect micro
amounts of NAD+ produced by the recycling. 1) The
concentration of alcohol and resazurin must be in excess to keep the
reaction velocity reaching to its maximum. 2) The concentration of
NAD+ and NADH must be lower than the
Km value to keep the enzymatic
reaction at its first order kinetics. 3) The amount of alcohol
dehydrogenase and diaphorase must be titered to the
concentration of NAD+ and NADH to match the basic
Michaelis-Menten’s equation of enzyme kinetics. To fulfill the first
requirement, we optimized the concentration of resazurin and found that
8 µmol/L satisfied both the excess substrate requirement and had
minimal quenching effect on the fluorescent product. To match the
requirement of the Michaelis-Menten kinetics for the formation of NADH
and NAD+, we titrated the ratio of alcohol
dehydrogenase and diaphorase. The widest detection linear range
(0.1 100 nmol) was obtained when the ratio of the two enzymes
was 0.5 U to 0.0125 U. These concentrations were 20 to 1000 times lower
than that of NAD+ and NADH. The reaction
catalyzed by alkaline phosphatase was also carefully studied. To
optimize alkaline-phosphatase activity itself, we also studied the
optimal concentration of NADP+ for an efficient
production of NAD+. We found that both
NAD+ and the background were increased with an
increase in NADP+ and alkaline phosphatase. When
the concentration of NADP+ was 1 µmol/L and
alkaline phosphatase 0.25 20 x
10-20 mol, a relatively high sensitivity with a
wide linear range and a low background were achieved.
It has been generally accepted that abnormal phosphorylation of tau is the first and most critical step for the formation of PHF and neurofibrillary tangles in AD brain.25,37 It is not known exactly how long it takes from abnormal phosphorylation of tau to the hallmark lesion of tangle formation in AD brain. However, various clinical and histopathological studies have suggested that AD is a chronic and progressive neurodegenerative disorder, and thus, the pathological processes might occur long before the appearance of clinical signs and symptoms. Therefore, early diagnosis of the disease is important both for establishing prevention and for evaluating the efficacy of therapeutic drugs. In this regard, the information provided in the present study is highly significant.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported in part by the Natural Science Foundation of China (grants 39925012, G1999054007, and 39870767), the Science and Technology Committee of China, the National Educational Committee of China, the National Institutes of Health (grants AG08076 and TW00703), and the New York State Office of Mental Retardation and Developmental Disabilities.
Accepted for publication January 2, 2002.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
(tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA 1986, 83:4913-4917This article has been cited by other articles:
 |
|
 |
|
  H. Zetterberg, M. Jonsson, A. Rasulzada, C. Popa, E. Styrud, M. A. Hietala, L. Rosengren, A. Wallin, and K. Blennow No neurochemical evidence for brain injury caused by heading in soccer Br. J. Sports Med., September 1, 2007; 41(9): 574 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Li, I. Sokal, J. F. Quinn, J. B. Leverenz, M. Brodey, G. D. Schellenberg, J. A. Kaye, M. A. Raskind, J. Zhang, E. R. Peskind, et al. CSF tau/A{beta}42 ratio for increased risk of mild cognitive impairment: A follow-up study Neurology, August 14, 2007; 69(7): 631 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zetterberg, M. A. Hietala, M. Jonsson, N. Andreasen, E. Styrud, I. Karlsson, A. Edman, C. Popa, A. Rasulzada, L.-O. Wahlund, et al. Neurochemical aftermath of amateur boxing. Arch Neurol, September 1, 2006; 63(9): 1277 - 1280. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hampel, K. Buerger, R. Zinkowski, S. J. Teipel, A. Goernitz, N. Andreasen, M. Sjoegren, J. DeBernardis, D. Kerkman, K. Ishiguro, et al. Measurement of Phosphorylated Tau Epitopes in the Differential Diagnosis of Alzheimer Disease: A Comparative Cerebrospinal Fluid Study Arch Gen Psychiatry, January 1, 2004; 61(1): 95 - 102. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
