Conceived and designed the experiments: PO PS SL MO BF. Performed the experiments: PO PS SJ HAK MO ACL BF. Analyzed the data: PO PS MO BF. Wrote the paper: PO BF.
The authors PO and BF are employees of Boehringer Ingelheim Pharma GmbH & Co. KG. Funding does not alter the authors' adherence to all the PLoS ONE polices on sharing data and material.
The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD).
Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (−70%) and cGMP (−55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99).
We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.
The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers. Synthesis by adenylate and guanylate cyclases and degradation by phosphodiesterases (PDEs) regulate the concentrations of cAMP and cGMP
In the brain, cAMP and cGMP signaling is involved in a multitude of mechanisms in neurons, astrocytes, oligodendrocytes and microglia. Examples are signal transduction in synapses, communication between neurons and glia cells or inflammatory processes
To date, clinical or even preclinical biomarkers for neurodegenerative diseases such as PD, ALS, CJD are desirable and in the focus of biomedical research
The aim of the present study is to investigate cAMP and cGMP in CSF of PD, PDD, ALS, CJD and control patients. The analysis of cAMP and cGMP in CSF is carried out with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). To evaluate the suitability of cAMP and cGMP as biomarkers we use receiver operating characteristic (ROC) curves and the Youden index
All patients included in this study attended to the general outpatient clinic, the outpatient memory clinic, the outpatient clinic for motoneuron diseases or the surveillance unit for transmissible spongiform encephalopathies (University of Göttingen and University of Ulm, Departments of Neurology). The Ethics Committees of the Universities Göttingen and Ulm approved the collection and analysis of CSF samples and specifically approved this study (Ulm). Samples of CJD patients were obtained from the surveillance and therapy trail
Diagnosis | Gender (m/f) | Age (years) | Disease related characteristics | |
|
|
|
||
CON | 4/5 | 55.4±18.3 | - | - |
PD | 6/5 | 69.6±9.0 | 1.9±0.7 |
27.4±6.4 |
PDD | 2/6 | 77.9±9.0 | 3.2±1.4 | 19.3±7.5 |
0.005 | 0.06 | 0.04 | ||
|
|
|||
CON | 5/9 | 49.0±16.8 | - | |
ALS | 9/5 | 61.4±11.0 | S = 8, B = 4, S+B = 2 | |
0.06 | ||||
|
|
|
||
CON | 6/5 | 66.4±11.8 | not done | 300±237 |
CJD | 6/9 | 60.3±12.9 | 10188±8477 | |
0.32 | 0.001 |
Data are means ± SD, Data are missing for.
three,
six and.
one patients.
CON: control patients, m: male, f: female, S: spinal, B: bulbar, S+B: spinal and bulbar, PRNP: prion protein gene, M: methionine, V: valine.
The group consisted of 14 patients diagnosed with ALS.
We investigated different control groups for the three neurodegenerative diseases to account for age differences.
The control patients had the following diagnosis: polymyositis (two patients), diabetes mellitus (two patients), amnesia, sinusitis. In three patients the lumbar puncture was carried out to exclude an acute or chronic inflammation.
The 14 patients in the control group had the following diagnoses: migraine with aura, episodic headache and symptomatic epilepsy, transient ischemic attack, borreliosis, anterior ischemic optic neuropathy (AION), PD, vestibular neuritis with depression and iron deficiency anemia, pseudotumor cerebri (PTC), migraine and polymyalgia rheumatica. For one of the control patients a lumbar puncture was carried out to exclude an acute or chronic inflammation.
The patients in the control group had the following diagnoses: migraine, vestibular neuritis, ALS, vertigo, epilepsy, polyneuropathy, subcortical vascular disease and ischemia, recessive transient attacks, lower body parkinsonism, neuritis retrobulbaris and for one case the lumbar puncture was carried out to exclude an acute or chronic inflammation.
Total tau protein concentrations in CSF samples of the CJD cohort were measured by a commercially available ELISA (Innogenetics, Gent, Belgium)
The CSF samples (35 µL) were mixed with 0.4 M perchloric acid (1∶1) and centrifuged at 20000× g for 10 min at 4°C to precipitate and remove proteins. The supernatant was analysed in duplicate by LC-MS/MS as described previously
Eluates were detected with an API 4000 triple quadrupole mass spectrometer (AB Sciex, Ontario, Canada) in the positive electrospray ionisation (ESI) mode by multiple reaction monitoring (MRM). The following transitions were used: 330.08 → 136.10 (cAMP) and 346.15 → 152.10 (cGMP). The injection volume was 25 µL.
Standard solutions were prepared in a mixture (1∶1) of artificial CSF (aCSF, 147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 0.85 mM MgCl2, and 1 mM Na2HPO4, pH 7.0–7.4) and 0.4 M perchloric acid. The stable isotope-labelled internal standards 13C5-cAMP (transition: 335.15 → 136.20) and 15N5-cGMP (transition: 351.03 → 157.00) were added to the standard solutions and CSF samples in a concentration of 100 nM to account for matrix effects and variations in ionisation.
A CSF sample was divided into 100 µL aliquots and frozen at −80°C to investigate the stability of cAMP and cGMP. Afterwards the aliquots were either stored at 4°C or at room temperature (RT) for the indicated time (see
Stability of cAMP and cGMP in human CSF after different handling and storage conditions. A CSF sample was splitted and subjected to the indicated procedures before being measured by LC-MS/MS in triplicate. Data were analysed by a one-way ANOVA followed by Dunnett's multiple comparison tests to compare the different groups against the control. Data are means ± SD,
All statistical analyses were carried out with GraphPad Prism 5.03 (La Jolla, USA). The data of the stability measurement were analysed by a one-way ANOVA and compared with the control sample by Dunnett's multiple comparison test. The cAMP and cGMP concentrations of the different groups of patients were compared by a Mann-Whitney or Kruskal-Wallis test. Spearman's rank correlation coefficient (r) was calculated for cAMP or cGMP with the Hoehn & Yahr score, MMSE, age (PD and PDD), tau protein as well as survival (CJD). A
Different storage and handling conditions during and after CSF collection could affect the results. We checked the stability of cAMP and cGMP in CSF under diverse conditions to reduce the risk of false positive or negative results.
The concentrations measured in the differently treated CSF samples were all within the range of ± 1 SD of the control sample (
We measured the concentrations of cAMP and cGMP in CSF samples of patients suffering from PD or PDD and control patients (CON) (
CSF concentrations of cAMP (A) and cGMP (B) in cases with Parkinson's disease (PD, n = 11), PD dementia (PDD, n = 8) and control patients (CON, n = 9) measured by LC-MS/MS. Data are means ± SEM,
A correlation analysis of the measured CSF concentrations in PD and PDD patients revealed no correlation between cAMP or cGMP and the Hoehn & Yahr score (
The measurement of the concentrations of cAMP and cGMP in CSF of ALS and control patients yielded mean values of 10.93 nM (CON) and 10.81 nM (ALS) for cAMP and 2.00 nM (CON) and 1.64 nM (ALS) for cGMP (
CSF concentrations of cAMP (A) and cGMP (B) in cases with amyotrophic lateral sclerosis (ALS, n = 14) and control patients (CON, n = 14) measured by LC-MS/MS. Data are means ± SEM,
The LC-MS/MS analysis of cAMP and cGMP in CSF of patients with CJD showed a marked reduction of cAMP of about 70% compared with controls (
CSF concentrations of cAMP (A) and cGMP (B) in cases with Creutzfeldt-Jakob disease (CJD, n = 15) and control patients (CON, n = 11) measured by LC-MS/MS. Data are means ± SEM, **
Since tau protein is suggested for the diagnosis of CJD we looked for a correlation with the cyclic nucleotides
Correlation of tau protein with cAMP (A) or cGMP (B) concentrations in CSF of Creutzfeldt-Jakob disease (CJD) patients. Spearman's rank correlation coefficient (r) and the respective
We further investigated, whether cAMP and cGMP concentrations correlated with the survival of the CJD patients. The patients had a survival time of 50.9±25.1 d (mean ± SEM) after lumbar puncture. There was no correlation with cAMP (
We calculated ROC curves to evaluate the diagnostic suitability of the cyclic nucleotides in CJD. ROC curves are depicted in
Receiver operating characteristic (ROC) curves of cAMP, cGMP and tau protein concentrations in CSF and combinations of them (cAMP×cGMP, tau/cAMP) for the differentiation of Creutzfeldt-Jakob disease (CJD) and control patients. Values for the area under the curve (AUC) and the best combinations of sensitivity and specificity are listed in
Parameter | cAMP | cGMP | cAMP×cGMP | Tau protein | Tau/cAMP |
AUC | 0.8606 | 0.8545 | 0.9152 | 0.9758 | 0.9879 |
0.002 | 0.002 | 0.001 | 0.001 | 0.001 | |
Cut-off |
<10.44 nM | <0.96 nM | <5.92 | >977 pg/mL | >174.7 |
Sensitivity (%) | 100 | 66.7 | 80.0 | 93.3 | 93.3 |
Specificity (%) | 63.6 | 100 | 90.9 | 100 | 100 |
AUC: area under the curve.
The cut-off was calculated using the Youden index
The ratio of tau/cAMP also resulted in an increase of the AUC. Cyclic GMP had the same effect (data not shown).
In this study, we investigated the concentrations of the cyclic nucleotides cAMP and cGMP in CSF of patients suffering from PD, PDD, ALS or CJD in comparison with controls. The measured values for cAMP and cGMP in the control groups agree with previously reported data showing concentrations in the range of 8–14 nM (cAMP) and 2–3 nM (cGMP)
Both nucleotides cAMP and cGMP are stable in human CSF during different handling and storage conditions as shown in
The impact of the cyclic nucleotides cAMP and cGMP in CSF of PD cases has been questioned for several years
In contrast to PD, there are no studies of the cyclic nucleotide concentrations in CSF of PDD patients. Similar to the observation in PD patients without dementia we did not find differences in PDD cases. There are reports of alterations in other types of dementia but the findings are not consistent. The discrepancies could be ascribed to distinct primary disorders causing the dementia
Two studies investigated CSF cGMP concentrations in ALS patients so far with conflicting results. Ilzecka (2004) showed a 50% decrease of cGMP in CSF of ALS cases but an earlier study by Ikeda et al. (1995) observed no difference
For the first time we investigated concentrations of cAMP and cGMP in CSF of Creutzfeldt-Jakob disease patients. Since there is a substantial degeneration of the whole brain in prion diseases in a relatively short time-span
In our study, the CJD patients distinguish from PD and ALS cases by the reduction of cAMP and cGMP concentrations in CSF. Therefore, we evaluated the suitability of cAMP and cGMP as biomarkers for CJD. The ROC analysis showed that both cyclic nucleotides have the potential for a diagnostic marker. With a sensitivity of 100% and specificity of 63.6% for cAMP and a sensitivity of 66.7% and specificity of 100% for cGMP they do not reach the potency of other CJD biomarkers, e.g. tau protein, 14-3-3 protein or S-100 protein
In contrast to cGMP, there is a significant correlation between tau protein and cAMP concentrations. The combination of tau with cAMP in a single ratio tau/cAMP again resulted in an improvement of the diagnostic potency although sensitivity and specificity where similar. We assume that the potential of the combination of tau protein and cAMP is underestimated in our study since the potency of tau protein itself is very high in our cohort. Using cAMP in this ratio seems to be more useful than cGMP because of the more pronounced decrease of cAMP in CJD patients.
Although the diagnostic potency of cAMP and cGMP alone or in combination is lower than for other recently established potential biomarkers of CJD such as ERK2 or ubiquitin, there are still some advantages of the cyclic nucleotides
Immunological assays, which are often preferred in the clinic, are commercially available for the determination of cAMP and cGMP if LC-MS/MS analysis is not possible. They enable a simple and fast analysis without involving expensive equipment. However, Zhang and colleagues (2009) showed the advantage of LC-MS/MS over enzyme based immuno assays by comparing a competitive enzyme immunoassay (EIA) for the analysis of cGMP in plasma samples and a LC-MS/MS assay for the same analytes. The LC-MS/MS method showed a better precision and accuracy as well as reduced matrix effects
The combination of cAMP and cGMP or tau protein and cAMP is a good example for the combination of different markers to obtain a higher diagnostic potency. This is important since biomarkers related to the pathological process are not always available. Hence, “secondary” markers that indicate downstream effects of the pathological events have to be used. However, frequently they lack specificity for the disease as described above for tau protein. Such weaknesses could be solved or reduced by combinations of biomarkers. In this context, LC-MS/MS analysis is a beneficial tool as shown in the present study. It allows the simultaneous measurement of several analytes in a single run.
In conclusion, we showed reduced concentrations of cAMP and cGMP in CSF of CJD patients but not in PD, PDD or ALS. This may be owing to the widespread brain atrophy in CJD compared with the localised neurodegeneration in the other diseases. Especially in combination, cAMP and cGMP have a diagnostic potential for CJD. In combination with tau protein, cAMP led to a further improvement of this marker. Since there are already fast and reliable immunological assays for cAMP and cGMP available the cyclic nucleotides can easily be included into routine analysis and may improve the diagnosis of CJD if LC-MS/MS is not applicable. The measurement of cAMP and cGMP CSF concentrations may be useful to monitor disease progression and therapy control. Our study also highlighted the advantages of LC-MS/MS for the combination of different biomarkers.
We would like to thank Ralf Kiesling, Stefan Aßfalg and Daniel Haerle (Boehringer Ingelheim Pharma GmbH & Co. KG, Department of Drug Metabolism & Pharmacokinetics) for the synthesis of the stable isotope-labelled internal standards.