3,4-Dihydroxyphenylglycol levels separate multiple system atrophy from Parkinson disease with orthostatic hypotension

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Goldstein, Patti Sullivan, Courtney Holmes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6675849/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Sep, 2025 Read the published version in Clinical Autonomic Research → Version 1 posted 4 You are reading this latest preprint version Abstract Background The autonomic synucleinopathy multiple system atrophy (MSA) can be difficult to distinguish clinically from Parkinson disease with orthostatic hypotension (PD + OH). 18 F-Dopamine positron emission tomography separates these conditions based on cardiac noradrenergic deficiency in PD + OH and not in MSA but is available only at the NIH Clinical Center. 3,4-Dihydroxyphenylglycol (DHPG) is the main neuronal metabolite of norepinephrine. This retrospective observational study examined whether DHPG levels in cerebrospinal fluid (CSF) or plasma differentiate MSA from PD + OH. Methods We reviewed CSF and plasma neurochemical data from all patients referred for evaluation at the NIH Clinical Center between 1995 and 2024 for chronic autonomic failure or parkinsonism. A concurrently studied comparison group were healthy volunteers or patients with orthostatic intolerance. Results CSF DHPG was decreased in MSA (N = 46, p < 0.0001) compared to the controls but also tended to be decreased in PD + OH (N = 16, p = 0.0598). Antecubital venous plasma DHPG was decreased in PD + OH (N = 40, p < 0.0001) but also in MSA (N = 59, p = 0.0458). CSF/plasma concentration ratios of DHPG were lower in MSA than in PD + OH (p < 0.0001). Cardiac arteriovenous increments in plasma DHPG and cardiac norepinephrine spillovers were strikingly decreased in PD + OH (N = 6) and were lower than in MSA (N = 20, p < 0.0001 each). Combining cardiac arteriovenous increments in plasma DHPG with norepinephrine spillovers completely separated PD + OH from MSA. Conclusions CSF/plasma ratios of DHPG, cardiac venous-arterial differences in plasma DHPG, and cardiac norepinephrine spillovers separate MSA from PD + OH. From our results we propose that biomarker combinations involving DHPG in biofluids may enable a pathophysiological differential diagnosis of MSA vs. PD + OH. DHPG Parkinson multiple system atrophy orthostatic hypotension sympathetic Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Multiple system atrophy (MSA) and Parkinson disease (PD) are synucleinopathies involving intra-cytoplasmic deposition of the protein alpha-synuclein (a-syn). In MSA the deposits are in glial cytoplasmic inclusions [ 47 ], whereas in PD a-syn is deposited in Lewy bodies [ 44 ]. MSA and PD often entail clinical manifestations of chronic autonomic failure [ 33 ] and are classified as autonomic synucleinopathies. Neurogenic orthostatic hypotension (nOH) occurs in a substantial minority of PD [ 46 ] and in most MSA [ 48 ] patients. It is difficult to distinguish PD with OH (PD + OH) from MSA by clinical criteria alone, especially early in the disease course. There is an ongoing need for pathophysiologically relevant biomarkers that can differentiate the two conditions. 18 F-Dopamine ( 18 F-DA) positron emission tomography (PET) to identify cardiac noradrenergic deficiency efficiently separates MSA from PD + OH [ 37 ], but for many years this testing modality has been available only at the NIH Clinical Center. Moreover, 18 F-DA PET is investigational, expensive, and involves radioactivity exposure. Several recent studies have reported on CSF a-syn seeding activity [ 40 , 41 ] or a-syn deposition in skin biopsies [ 3 , 4 , 8 ], but so far it is unclear how well these tests separate MSA and PD + OH from each other and from other autonomic disorders. Both MSA and PD + OH feature central noradrenergic deficiency, as indicated in vivo by decreased cerebrospinal fluid (CSF) levels of norepinephrine (NE) and its metabolites [ 23 ] and post-mortem by decreased putamen tissue NE contents [ 22 ]. In the periphery, orthostatic increments in plasma NE are attenuated in both MSA [ 29 ] and PD + OH [ 12 ], reflecting baroreflex-sympathoneural failure. MSA and PD + OH differ in the occurrence of sympathetic noradrenergic deficiency [ 50 ], especially in the heart. In vivo and post-mortem data have consistently indicated a substantial cardiac sympathetic noradrenergic lesion in PD + OH and intact sympathetic innervation in most (but not all) patients with MSA [ 2 , 21 ]. The difference in peripheral abnormalities despite similar central noradrenergic abnormalities provided the theoretical backdrop for the present study. 3,4-Dihydroxyphenylglycol (DHPG) is the main neuronal metabolite of NE. Based on the above considerations, in this retrospective observational study we hypothesized that CSF/plasma ratios of NE and DHPG, cardiac NE spillovers, and cardiac venous-arterial differences in plasma DHPG would distinguish PD + OH from MSA. METHODS Study Subjects All the participants in this study gave written informed consent before any research procedures were done. The protocols were approved by the Institutional Review Board of the National Institute of Neurological Disorders and Stroke (NINDS) or of the National Institutes of Health (NIH). All the patients had been referred to the Autonomic Medicine Section (formerly Clinical Neurocardiology Section) of the Division of Intramural Research of the NINDS for known or suspected autonomic dysfunction and were studied at the NIH Clinical Center. Accrual was by referral only; there was no recruitment by advertisement. The presence or absence of neurogenic OH (nOH) was determined based on beat-to-beat blood pressure responses to the Valsalva maneuver or orthostatic fractional increments in plasma NE levels [ 16 , 26 ]. Patients were stratified into PD + OH or MSA groups based on previously published consensus statements [ 7 , 9 , 10 , 32 ]. We also used the UK Brain Bank criteria for PD [ 28 ], with an important exception. According to the UK Brain Bank criteria, early, prominent autonomic involvement is exclusionary for diagnosing PD. Findings by our group [ 11 , 14 , 17 ] and others [ 38 ] that OH can be an early finding in PD disagree with this assertion. Most of the patients underwent cardiac sympathetic neuroimaging by 18 F-DA positron emission tomography (PET), which efficiently separates PD + OH from MSA, in that virtually all patients with PD + OH have low myocardial 18 F- DA-derived radioactivity, whereas most patients with MSA have normal radioactivity [ 21 , 37 ]. Blood sampling With the subject supine, an intravenous catheter was placed percutaneously in an arm vein, usually an antecubital vein. A 3-way stopcock was attached to the hub of the catheter. Normal saline was infused at a slow rate to keep the vein open. After the subject had been supine for at least 15 minutes, about 1 mL of blood was drawn through the stopcock and discarded. About 5 mL of blood was then sampled and placed in ice. The blood was centrifuged in a refrigerated centrifuge and the plasma transferred to a plastic cryotube and stored at -80 o C until thawed for assay. Lumbar puncture Lumbar puncture was performed under fluoroscopic guidance by a Board-certified neuroradiologist or by a neuroradiology post-doctoral fellow. Aliquots of 1 mL of CSF were frozen immediately in dry ice and kept frozen at -80 o C until thawed for assay. In most cases the 6th aliquot was assayed for catechols. Right heart cardiac catheterization Right heart catheterization was performed by a team led by a Board-certified cardiologist who was credentialed at the NIH Clinical Center. A left brachial arterial catheter was placed for monitoring blood pressure and obtaining blood samples. The right internal jugular vein was cannulated percutaneously after anesthesia of the overlying skin, and a plastic catheter was advanced into the great cardiac vein or coronary sinus. The configuration of the catheter was the reverse of a Swan-Ganz catheter; saline injectate at room temperature was administered distal rather proximal to the thermistor, enabling measurement of coronary sinus blood flow by thermodilution. Tracer-labeled NE was infused continuously into a systemic vein. After at least 20 minutes of supine rest, an arterial and a cardiac venous blood sample were obtained approximately simultaneously. The present study relied on cardiac catheterization data from a previously published study [ 13 ]. Relationships among arm and cardiac venous-arterial differences in plasma DHPG and cardiac NE spillover in MSA vs. PD + OH have not been reported previously. 18 F-Dopamine PET Most of the patients underwent cardiac sympathetic neuroimaging by 18 F-DA PET as reported previously [ 13 ]. Briefly, 1 mCi of 18 F-DA was infused intravenously over 3 minutes. Interventricular septal myocardial 18 F-DA-derived radioactivity was measured in the 5’ dynamic frame with midpoint about 8’ after initiation of administration of the tracer. Radioactivity concentrations in nCi/cc were adjusted for the dose injected per kg, so that the radioactivity concentrations were in units of nCi-kg/cc-mCi. Previous studies have not examined relationships of CSF/plasma ratios of DHPG levels to cardiac 18 F-DA-derived radioactivity. Catechol assay Plasma and CSF were assayed by batch alumina extraction followed by liquid chromatography with series electrochemical detection as described previously by our group [ 15 , 27 ]. Avoidance of biases Personnel conducting neurochemical assays or analyses of PET images were blinded as to the other clinical laboratory results until the data were tabulated. Data Analysis and Statistics Cardiac NE spillover (in pmol/min) was calculated as described previously [ 6 ] using the tracer dilution principle. Briefly, a tracer amount of 3 H-NE was infused to a steady-state (≥ 20 minutes), and cardiac NE Spillover calculated according to the equation NE Spillover = Q * (NEv – NEa) * (SAa / SAv) where Q = coronary sinus plasma flow (in mL/min), NEv = cardiac venous NE concentration (in pmol/mL), NEa = arterial NE concentration (in pmol/mL), SAa = specific activity of 3 H-NE in arterial plasma, and SAv = specific activity of 3 H-NE in cardiac venous plasma. For statistical analyses and graphics GraphPad Prism 9 for Mac (GraphPad Software, Boston, MA) was used. Mean values in the PD + OH, MSA, and Control groups were compared by factorial analyses of variance with Tukey’s post-hoc test. Pearson correlation coefficients were calculated for scatterplots. Receiver operating characteristic (ROC) curves were generated for evaluating the efficiency of CSF/plasma ratios of DHPG to separate MSA from PD + OH and from controls. Frequencies were analyzed by Fisher’s exact test. A p value less than 0.05 defined statistical significance. RESULTS Plasma catechols data were reviewed from 212 participants (66 MSA, 42 PD + OH, 104 controls) and CSF catechols data from 152 participants (68 MSA, 31 PD + OH, 53 controls). Demographic data for the MSA, PD + OH, and Control groups are in the Supplementary Data Workbook. All the patients were off levodopa/carbidopa as documented by CSF or plasma DOPA levels within the respective normal ranges. Compared to values in the Control group, in the MSA group CSF DOPA, DOPAC, NE, and DHPG were decreased (Fig. 1 A- 1 D), while in the PD + OH group CSF DOPAC was decreased but not CSF DOPA, NE, or DHPG. Antecubital venous plasma NE was decreased in PD + OH (Fig. 1 E) but not in MSA. Plasma DHPG was decreased in both groups (Fig. 1 F) but more clearly so in PD + OH. The groups did not differ in arm venous-arterial differences in plasma DHPG levels (Fig. 1 G). CSF/plasma ratios of both DHPG (Fig. 2 A) and NE (Fig. 2 B) were lower in MSA than in PD + OH, with the group difference most noticeable for CSF/plasma ratios of DHPG. (For analyzing data for CSF/plasma ratios of NE, data from 2 PD + OH patients with outlying data were excluded, and arterial DHPG was used for 3 subjects because arm venous DHPG data were unavailable.) CSF/plasma ratios of DHPG and NE considered together separated a subgroup of MSA patients from a subgroup of PD + OH patients (MSA 0/16 = 0% within the control range (pink rectangle in Fig. 2 C, PD 11/16 = 69% within the control range, p < 0.0001 by Fisher’s exact test). ROC analyses for 26 MSA vs. 26 PD + OH patients showed a ROC area of 0.84763 (95% confidence interval 0.74085–0.95442, p < 0.0001; Fig. 2 D). For 26 MSA patients vs. 28 controls the ROC area was 0.82486 (95% confidence interval 0.71106–0.93867, p < 0.0001; Fig. 2 E). Cardiac 18 F-DA-derived radioactivity efficiently separated the MSA from the PD + OH groups (Fig. 3 A), with the radioactivity decreased from control in PD + OH and increased from control in MSA. Similarly, cardiac NE spillovers were decreased from control in PD + OH and increased from control in MSA (Fig. 3 B). Cardiac arteriovenous increments in plasma DHPG were strikingly decreased in PD + OH (Fig. 3 C) and tended to be decreased in MSA. MSA and PD + OH were highly efficiently separated when individual values for CSF/plasma ratios of DHPG were expressed as a function of cardiac 18 F-DA-derived radioactivity (blue and pink rectangles in Fig. 3 D) and when cardiac arteriovenous increments in plasma DHPG were expressed as a function of cardiac NE spillovers (blue and pink rectangles in Fig. 3 E). DISCUSSION There is a longstanding need for pathophysiologically relevant biomarkers that can separate MSA from PD + OH. In the present retrospective analysis we obtained evidence that CSF/plasma ratios of DHPG, CSF/plasma ratios of NE, cardiac arteriovenous increments in plasma DHPG, cardiac NE spillovers, and cardiac 18 F-DA-derived radioactivity all distinguish MSA from PD + OH. The following discussion explains why these findings make sense from a pathophysiological point of view. CSF DHPG probably mainly reflects central neural stores of NE, which are depleted in both PD + OH and MSA [ 15 , 24 ]. In the periphery, however, there is sympathetic noradrenergic deficiency in PD + OH [ 43 ], whereas in MSA sympathetic noradrenergic innervation and function generally are intact [ 20 , 42 ]. Accordingly, one would expect that CSF/plasma ratios of DHPG and NE would be lower in MSA than in PD + OH. When CSF/plasma ratios of DHPG and NE were considered together, none of the MSA patients had values for both variables within the control range, whereas most of the PD + OH patients did. These data suggest that the finding of low CSF/plasma ratios of DHPG and NE offers a positive biomarker for MSA compared to PD + OH. The relative preservation of peripheral DHPG levels in MSA despite central NE depletion likely reflects intact peripheral sympathetic innervation and normal intra-neuronal enzymatic metabolism of cytoplasmic NE, in contrast with denervation and a shift from vesicular sequestration to enzymatic metabolism in PD + OH [ 18 , 25 ]. The sympathetic noradrenergic lesion in PD + OH is especially prominent in the heart [ 35 ]. Thus, reported previously that patients with PD + OH have markedly decreased cardiac NE spillovers even when total body NE spillovers are within normal limits [ 13 ]. We have also reported previously that PD + OH entails attenuated cardiac arteriovenous increments in plasma DHPG levels [ 13 ]. In this study we found that combining cardiac NE spillovers with cardiac arteriovenous increments in plasma DHPG levels completely separated MSA from PD + OH, whereas arm arteriovenous increments in plasma DHPG were ineffective in this regard. The present neurochemical and sympathetic neuroimaging findings add to growing evidence from cross-sectional studies that one can separate groups of patients with MSA or PD + OH by biomarkers. Other biomarkers include measures of olfactory dysfunction [ 39 ], diffusion tensor magnetic resonance imaging [ 34 ], a-syn seeding activity [ 41 ], a-syn deposition in skin biopsies [ 3 ], and CSF neurofilament light chain [ 1 ]. The relative efficiencies of these and other modalities, alone and especially in combination, for distinguishing MSA from PD + OH remain to be assessed. Overall, our data provide new support for DHPG measurements as a practical and potentially scalable clinical test for distinguishing MSA from PD + OH. Even without cardiac catheterization, the CSF/plasma DHPG ratio alone yields respectable separation between MSA and PD + OH in ROC analyses, suggesting potential utility in academic autonomic medicine centers. Limitations The participants in this study were highly selected and studied at a single site, and they agreed to undergo comprehensive testing without the presumption of personal benefit. Generalizability to the overall population of LBD patients is unknown. Relatively few groups in the United States carry out right heart catheterizations and arterial blood sampling, and none measure cardiac NE spillover, which requires both infusion of tracer-labeled NE and means to quantify coronary sinus blood flow. The present study suggests that cardiac arteriovenous increments in plasma DHPG may be sufficient to separate MSA from PD + OH in individual patients. This seems within the capability of cardiology investigators who evaluate patients with neurocardiological disorders at academic centers [ 5 , 31 , 45 ]. Commercial laboratories do not currently offer DHPG assays, and liquid chromatography with electrochemical detection, which has been a standard method used for many years [ 27 ], is becoming impractical because of the lack of availability of electrodes suitable for series electrochemical detection. Perhaps the present results will help convince laboratories that assay catecholamines by liquid chromatography with tandem mass spectroscopy to add DHPG to their repertoires. Implications This report introduces neurochemical biomarkers that separate PD + OH from MSA based on CSF and plasma DHPG levels. Prospective studies are needed to determine whether these biomarkers predict the trajectory of phenoconversion from clinically defined pure autonomic failure to central synucleinopathies [ 19 , 36 , 39 ]. Neurochemical biomarkers assessed in combination with other modalities such as skin biopsies with immunofluorescence confocal microscopy [ 30 ] or a-syn seed amplification assays [ 49 ] may lead to a multimodal framework that would enable diagnosis by convergence. Declarations ACKNOWLEDGEMENTS The research reported here was supported (in part) by the research Division of Intramural Research, National Institutes of Health (NINDS). Financial support: Division of Intramural Research, NINDS, NIH. CONFLICTS OF INTEREST: The authors have no conflicts of interest to disclose. AUTHOR CONTRIBUTIONS: DSG: Study concept, data analysis, manuscript writing PS: Data acquisition, data analysis, manuscript editing CH: Data acquisition, data analysis, manuscript editing References Abdo WF, Bloem BR, Van Geel WJ, Esselink RA, Verbeek MM (2007) CSF neurofilament light chain and tau differentiate multiple system atrophy from Parkinson's disease. Neurobiol Aging 28:742-747 Cook GA, Sullivan P, Holmes C, Goldstein DS (2014) Cardiac sympathetic denervation without Lewy bodies in a case of multiple system atrophy. Parkinsonism Relat. 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Med. 296:293-297 Supplementary Files DATADICTIONARYFORSUPPLEMENTARYDATAWORKBOOK.docx STROBEChecklist.docx Cite Share Download PDF Status: Published Journal Publication published 17 Sep, 2025 Read the published version in Clinical Autonomic Research → Version 1 posted Reviewers agreed at journal 26 May, 2025 Reviewers invited by journal 19 May, 2025 Editor assigned by journal 18 May, 2025 First submitted to journal 15 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6675849","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":458905613,"identity":"0ac22c53-e26b-4fa5-8521-45437faec539","order_by":0,"name":"David S. Goldstein","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYFAC5gYJIMnDwJB8gCGBgRnIZmwjoIURqCUBpCUtgTQtQJBjALITxGLDq0HevbHx5s8fNjLm7DnfHjzcYW3PIN3c9oDhl01iAw4thmcONlvzJKTxWPa83W6QeCY9sUHmYLsBY18abi0zEtukGRIO8xjcyN0mkdh2OIEBSEow9hw2xuUwkBbJHwn/gVpynoG02BPUIg9SwJNwAKSFDaQFGBpAEYYfh+VwaTHgAfklLZnH4MwzM6DidKB6EGpIw6lFvr354M0fNnb2BseTn0n+bLO255dIfybx4Y8ND05bDqCLgCMlEU9syjdgF/+DW8soGAWjYBSMOAAAFl5XqvNQKPkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5709-9940","institution":"National Institute of Neurological Disorders and Stroke Intramural Research Program","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"S.","lastName":"Goldstein","suffix":""},{"id":458905614,"identity":"4b6ff308-7ea8-44ef-b33f-40011596ef2e","order_by":1,"name":"Patti Sullivan","email":"","orcid":"","institution":"National Institute of Neurological Disorders and Stroke Division of Intramural Research","correspondingAuthor":false,"prefix":"","firstName":"Patti","middleName":"","lastName":"Sullivan","suffix":""},{"id":458905615,"identity":"936476b5-e436-453a-a6bd-d8368f98c1e2","order_by":2,"name":"Courtney Holmes","email":"","orcid":"","institution":"National Institute of Neurological Disorders and Stroke Division of Intramural Research","correspondingAuthor":false,"prefix":"","firstName":"Courtney","middleName":"","lastName":"Holmes","suffix":""}],"badges":[],"createdAt":"2025-05-15 23:14:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6675849/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6675849/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10286-025-01150-8","type":"published","date":"2025-09-17T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83280631,"identity":"e676419c-5fa0-4c68-addc-3dd333d3e7c9","added_by":"auto","created_at":"2025-05-22 10:12:13","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":484573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIndividual and mean (± SEM) values for cerebrospinal fluid (CSF) and plasma catechols in patients with Parkinson disease and orthostatic hypotension (PD+OH, red), multiple system atrophy (MSA, blue), and control subjects (Control, gray)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) CSF DOPA; (B) CSF 3,4-dihydroxyphenylacetic acid (DOPAC); (C) CSF norepinephrine (NE); (D) CSF 3,4-dihydroxyphenylglycol (DHPG); (E) plasma NE during supine rest; (F) plasma DHPG during supine rest; (G) Arm venous-arterial (V-A) difference in plasma DHPG. Numbers in italics are p values for group comparisons by Tukey’s test. In MSA but not in PD+OH CSF DOPA is decreased compared to controls. CSF DOPAC is decreased in both MSA and PD+OH. CSF NE is decreased in MSA but not in PD+OH. CSF DHPG is decreased in MSA and tends to be decreased in PD+OH. Plasma NE is decreased in PD+OH but not in MSA. Plasma DHPG is decreased in both MSA and PD+OH, moreso in PD+OH. The groups do not differ in venous-arterial differences in arm DHPG.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6675849/v1/6dd0af12aa12adecb9f58f75.jpeg"},{"id":83280942,"identity":"fa57bbcb-c5ac-494a-989c-0f91e2605e96","added_by":"auto","created_at":"2025-05-22 10:20:13","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCSF/plasma ratios in multiple system atrophy (MSA, blue), Parkinson disease with orthostatic hypotension (PD+OH, red), and control subjects (gray).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) CSF/plasma ratios of 3,4-dihydroxyphenylglycol (DHPG); (B) CSF/plasma ratios of norepinephrine (NE); (C) scatterplot relating individual data for CSF/plasma ratios of DHPG to data for CSF/plasma ratios of NE; (D) Receiver Operating Characteristic (ROC) curves for CSF/plasma ratios of DHPG in MSA vs. PD+OH; (E) ROC curves for CSF/plasma ratios of DHPG in MSA vs. controls. In (C) blue and pink rectangles placed manually to highlight separation of MSA from PD+OH (p\u0026lt;0.0001 by Fisher’s exact test).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6675849/v1/f971a8da12647c62d3b21f75.jpeg"},{"id":83280632,"identity":"5cc2f790-a594-4273-9f55-7602ed774d9e","added_by":"auto","created_at":"2025-05-22 10:12:13","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":425313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiomarkers separating multiple system atrophy (MSA, blue) from Parkinson disease with orthostatic hypotension (PD+OH, red) and control subjects (gray).\u003c/strong\u003e(A) Interventricular septal myocardial \u003csup\u003e18\u003c/sup\u003eF-dopamine (\u003csup\u003e18\u003c/sup\u003eF-DA)-derived radioactivity; (B) cardiac norepinephrine (NE) spillover; (C) cardiac venous-arterial (V-A) difference in plasma 3,4-dihydroxyphenylglycol (DHPG); (D) CSF/plasma ratio of DHPG vs. cardiac \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity; (E) cardiac NE spillover vs. cardiac venous-arterial (V-A) difference in plasma DHPG. In (D) and (E) pink and blue rectangles placed manually to emphasize differences between MSA and PD+OH. In (A) note decreased \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity in PD+OH and increased radioactivity in MSA compared to controls and in (B) low cardiac NE spillovers in PD+OH and elevated spillovers in MSA. Combining CSF/plasma ratios of DHPG with cardiac \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity and combining cardiac NE spillovers with cardiac venous-arterial (V-A) difference in plasma DHPG efficiently separated MSA from PD+OH.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6675849/v1/15a02031e65188a2e13b5192.jpeg"},{"id":91889866,"identity":"eda89832-cb37-42e1-aa15-5c3d54d50a1d","added_by":"auto","created_at":"2025-09-22 16:02:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1983352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6675849/v1/b6434401-f19f-4d8e-b02e-b3c5d725a7ba.pdf"},{"id":83279591,"identity":"3a8cc48b-8474-42bb-957a-e2a71122ef78","added_by":"auto","created_at":"2025-05-22 10:04:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14545,"visible":true,"origin":"","legend":"","description":"","filename":"DATADICTIONARYFORSUPPLEMENTARYDATAWORKBOOK.docx","url":"https://assets-eu.researchsquare.com/files/rs-6675849/v1/04a07130089447edb9ef539b.docx"},{"id":83279599,"identity":"63ae89c8-6841-45b1-b66e-efbab5e82e14","added_by":"auto","created_at":"2025-05-22 10:04:13","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":36011,"visible":true,"origin":"","legend":"","description":"","filename":"STROBEChecklist.docx","url":"https://assets-eu.researchsquare.com/files/rs-6675849/v1/b8e6ccc5935a4551ae9bb439.docx"}],"financialInterests":"","formattedTitle":"3,4-Dihydroxyphenylglycol levels separate multiple system atrophy from Parkinson disease with orthostatic hypotension","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMultiple system atrophy (MSA) and Parkinson disease (PD) are synucleinopathies involving intra-cytoplasmic deposition of the protein alpha-synuclein (a-syn). In MSA the deposits are in glial cytoplasmic inclusions [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], whereas in PD a-syn is deposited in Lewy bodies [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. MSA and PD often entail clinical manifestations of chronic autonomic failure [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and are classified as autonomic synucleinopathies. Neurogenic orthostatic hypotension (nOH) occurs in a substantial minority of PD [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and in most MSA [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] patients.\u003c/p\u003e \u003cp\u003eIt is difficult to distinguish PD with OH (PD\u0026thinsp;+\u0026thinsp;OH) from MSA by clinical criteria alone, especially early in the disease course. There is an ongoing need for pathophysiologically relevant biomarkers that can differentiate the two conditions. \u003csup\u003e18\u003c/sup\u003eF-Dopamine (\u003csup\u003e18\u003c/sup\u003eF-DA) positron emission tomography (PET) to identify cardiac noradrenergic deficiency efficiently separates MSA from PD\u0026thinsp;+\u0026thinsp;OH [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], but for many years this testing modality has been available only at the NIH Clinical Center. Moreover, \u003csup\u003e18\u003c/sup\u003eF-DA PET is investigational, expensive, and involves radioactivity exposure. Several recent studies have reported on CSF a-syn seeding activity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] or a-syn deposition in skin biopsies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], but so far it is unclear how well these tests separate MSA and PD\u0026thinsp;+\u0026thinsp;OH from each other and from other autonomic disorders.\u003c/p\u003e \u003cp\u003eBoth MSA and PD\u0026thinsp;+\u0026thinsp;OH feature central noradrenergic deficiency, as indicated in vivo by decreased cerebrospinal fluid (CSF) levels of norepinephrine (NE) and its metabolites [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and post-mortem by decreased putamen tissue NE contents [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the periphery, orthostatic increments in plasma NE are attenuated in both MSA [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and PD\u0026thinsp;+\u0026thinsp;OH [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], reflecting baroreflex-sympathoneural failure.\u003c/p\u003e \u003cp\u003eMSA and PD\u0026thinsp;+\u0026thinsp;OH differ in the occurrence of sympathetic noradrenergic deficiency [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], especially in the heart. In vivo and post-mortem data have consistently indicated a substantial cardiac sympathetic noradrenergic lesion in PD\u0026thinsp;+\u0026thinsp;OH and intact sympathetic innervation in most (but not all) patients with MSA [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The difference in peripheral abnormalities despite similar central noradrenergic abnormalities provided the theoretical backdrop for the present study.\u003c/p\u003e \u003cp\u003e3,4-Dihydroxyphenylglycol (DHPG) is the main neuronal metabolite of NE. Based on the above considerations, in this retrospective observational study we hypothesized that CSF/plasma ratios of NE and DHPG, cardiac NE spillovers, and cardiac venous-arterial differences in plasma DHPG would distinguish PD\u0026thinsp;+\u0026thinsp;OH from MSA.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Subjects\u003c/h2\u003e \u003cp\u003e All the participants in this study gave written informed consent before any research procedures were done. The protocols were approved by the Institutional Review Board of the National Institute of Neurological Disorders and Stroke (NINDS) or of the National Institutes of Health (NIH). All the patients had been referred to the Autonomic Medicine Section (formerly Clinical Neurocardiology Section) of the Division of Intramural Research of the NINDS for known or suspected autonomic dysfunction and were studied at the NIH Clinical Center. Accrual was by referral only; there was no recruitment by advertisement.\u003c/p\u003e \u003cp\u003eThe presence or absence of neurogenic OH (nOH) was determined based on beat-to-beat blood pressure responses to the Valsalva maneuver or orthostatic fractional increments in plasma NE levels [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePatients were stratified into PD\u0026thinsp;+\u0026thinsp;OH or MSA groups based on previously published consensus statements [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We also used the UK Brain Bank criteria for PD [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with an important exception. According to the UK Brain Bank criteria, early, prominent autonomic involvement is exclusionary for diagnosing PD. Findings by our group [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and others [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] that OH can be an early finding in PD disagree with this assertion. Most of the patients underwent cardiac sympathetic neuroimaging by \u003csup\u003e18\u003c/sup\u003eF-DA positron emission tomography (PET), which efficiently separates PD\u0026thinsp;+\u0026thinsp;OH from MSA, in that virtually all patients with PD\u0026thinsp;+\u0026thinsp;OH have low myocardial \u003csup\u003e18\u003c/sup\u003eF- DA-derived radioactivity, whereas most patients with MSA have normal radioactivity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlood sampling\u003c/h3\u003e\n\u003cp\u003eWith the subject supine, an intravenous catheter was placed percutaneously in an arm vein, usually an antecubital vein. A 3-way stopcock was attached to the hub of the catheter. Normal saline was infused at a slow rate to keep the vein open. After the subject had been supine for at least 15 minutes, about 1 mL of blood was drawn through the stopcock and discarded. About 5 mL of blood was then sampled and placed in ice. The blood was centrifuged in a refrigerated centrifuge and the plasma transferred to a plastic cryotube and stored at -80 \u003csup\u003eo\u003c/sup\u003eC until thawed for assay.\u003c/p\u003e\n\u003ch3\u003eLumbar puncture\u003c/h3\u003e\n\u003cp\u003eLumbar puncture was performed under fluoroscopic guidance by a Board-certified neuroradiologist or by a neuroradiology post-doctoral fellow. Aliquots of 1 mL of CSF were frozen immediately in dry ice and kept frozen at -80 \u003csup\u003eo\u003c/sup\u003eC until thawed for assay. In most cases the 6th aliquot was assayed for catechols.\u003c/p\u003e\n\u003ch3\u003eRight heart cardiac catheterization\u003c/h3\u003e\n\u003cp\u003eRight heart catheterization was performed by a team led by a Board-certified cardiologist who was credentialed at the NIH Clinical Center. A left brachial arterial catheter was placed for monitoring blood pressure and obtaining blood samples. The right internal jugular vein was cannulated percutaneously after anesthesia of the overlying skin, and a plastic catheter was advanced into the great cardiac vein or coronary sinus. The configuration of the catheter was the reverse of a Swan-Ganz catheter; saline injectate at room temperature was administered distal rather proximal to the thermistor, enabling measurement of coronary sinus blood flow by thermodilution. Tracer-labeled NE was infused continuously into a systemic vein. After at least 20 minutes of supine rest, an arterial and a cardiac venous blood sample were obtained approximately simultaneously.\u003c/p\u003e \u003cp\u003eThe present study relied on cardiac catheterization data from a previously published study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Relationships among arm and cardiac venous-arterial differences in plasma DHPG and cardiac NE spillover in MSA vs. PD\u0026thinsp;+\u0026thinsp;OH have not been reported previously.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e18\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eF-Dopamine PET\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMost of the patients underwent cardiac sympathetic neuroimaging by \u003csup\u003e18\u003c/sup\u003eF-DA PET as reported previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, 1 mCi of \u003csup\u003e18\u003c/sup\u003eF-DA was infused intravenously over 3 minutes. Interventricular septal myocardial \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity was measured in the 5\u0026rsquo; dynamic frame with midpoint about 8\u0026rsquo; after initiation of administration of the tracer. Radioactivity concentrations in nCi/cc were adjusted for the dose injected per kg, so that the radioactivity concentrations were in units of nCi-kg/cc-mCi. Previous studies have not examined relationships of CSF/plasma ratios of DHPG levels to cardiac \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity.\u003c/p\u003e\n\u003ch3\u003eCatechol assay\u003c/h3\u003e\n\u003cp\u003ePlasma and CSF were assayed by batch alumina extraction followed by liquid chromatography with series electrochemical detection as described previously by our group [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAvoidance of biases\u003c/h2\u003e \u003cp\u003ePersonnel conducting neurochemical assays or analyses of PET images were blinded as to the other clinical laboratory results until the data were tabulated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData Analysis and Statistics\u003c/h3\u003e\n\u003cp\u003eCardiac NE spillover (in pmol/min) was calculated as described previously [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] using the tracer dilution principle. Briefly, a tracer amount of \u003csup\u003e3\u003c/sup\u003eH-NE was infused to a steady-state (\u0026ge;\u0026thinsp;20 minutes), and cardiac NE Spillover calculated according to the equation\u003c/p\u003e \u003cp\u003eNE Spillover\u0026thinsp;=\u0026thinsp;Q \u003csub\u003e*\u003c/sub\u003e (NEv \u0026ndash; NEa) \u003csub\u003e*\u003c/sub\u003e (SAa \u003csub\u003e/\u003c/sub\u003e SAv)\u003c/p\u003e \u003cp\u003ewhere Q\u0026thinsp;=\u0026thinsp;coronary sinus plasma flow (in mL/min), NEv\u0026thinsp;=\u0026thinsp;cardiac venous NE concentration (in pmol/mL), NEa\u0026thinsp;=\u0026thinsp;arterial NE concentration (in pmol/mL), SAa\u0026thinsp;=\u0026thinsp;specific activity of \u003csup\u003e3\u003c/sup\u003eH-NE in arterial plasma, and SAv\u0026thinsp;=\u0026thinsp;specific activity of \u003csup\u003e3\u003c/sup\u003eH-NE in cardiac venous plasma.\u003c/p\u003e \u003cp\u003eFor statistical analyses and graphics GraphPad Prism 9 for Mac (GraphPad Software, Boston, MA) was used.\u003c/p\u003e \u003cp\u003eMean values in the PD\u0026thinsp;+\u0026thinsp;OH, MSA, and Control groups were compared by factorial analyses of variance with Tukey\u0026rsquo;s post-hoc test. Pearson correlation coefficients were calculated for scatterplots. Receiver operating characteristic (ROC) curves were generated for evaluating the efficiency of CSF/plasma ratios of DHPG to separate MSA from PD\u0026thinsp;+\u0026thinsp;OH and from controls. Frequencies were analyzed by Fisher\u0026rsquo;s exact test. A p value less than 0.05 defined statistical significance.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003ePlasma catechols data were reviewed from 212 participants (66 MSA, 42 PD\u0026thinsp;+\u0026thinsp;OH, 104 controls) and CSF catechols data from 152 participants (68 MSA, 31 PD\u0026thinsp;+\u0026thinsp;OH, 53 controls). Demographic data for the MSA, PD\u0026thinsp;+\u0026thinsp;OH, and Control groups are in the Supplementary Data Workbook. All the patients were off levodopa/carbidopa as documented by CSF or plasma DOPA levels within the respective normal ranges.\u003c/p\u003e \u003cp\u003eCompared to values in the Control group, in the MSA group CSF DOPA, DOPAC, NE, and DHPG were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), while in the PD\u0026thinsp;+\u0026thinsp;OH group CSF DOPAC was decreased but not CSF DOPA, NE, or DHPG. Antecubital venous plasma NE was decreased in PD\u0026thinsp;+\u0026thinsp;OH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) but not in MSA. Plasma DHPG was decreased in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) but more clearly so in PD\u0026thinsp;+\u0026thinsp;OH. The groups did not differ in arm venous-arterial differences in plasma DHPG levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCSF/plasma ratios of both DHPG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and NE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) were lower in MSA than in PD\u0026thinsp;+\u0026thinsp;OH, with the group difference most noticeable for CSF/plasma ratios of DHPG. (For analyzing data for CSF/plasma ratios of NE, data from 2 PD\u0026thinsp;+\u0026thinsp;OH patients with outlying data were excluded, and arterial DHPG was used for 3 subjects because arm venous DHPG data were unavailable.) CSF/plasma ratios of DHPG and NE considered together separated a subgroup of MSA patients from a subgroup of PD\u0026thinsp;+\u0026thinsp;OH patients (MSA 0/16\u0026thinsp;=\u0026thinsp;0% within the control range (pink rectangle in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, PD 11/16\u0026thinsp;=\u0026thinsp;69% within the control range, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 by Fisher\u0026rsquo;s exact test). ROC analyses for 26 MSA vs. 26 PD\u0026thinsp;+\u0026thinsp;OH patients showed a ROC area of 0.84763 (95% confidence interval 0.74085\u0026ndash;0.95442, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). For 26 MSA patients vs. 28 controls the ROC area was 0.82486 (95% confidence interval 0.71106\u0026ndash;0.93867, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCardiac \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity efficiently separated the MSA from the PD\u0026thinsp;+\u0026thinsp;OH groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with the radioactivity decreased from control in PD\u0026thinsp;+\u0026thinsp;OH and increased from control in MSA. Similarly, cardiac NE spillovers were decreased from control in PD\u0026thinsp;+\u0026thinsp;OH and increased from control in MSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Cardiac arteriovenous increments in plasma DHPG were strikingly decreased in PD\u0026thinsp;+\u0026thinsp;OH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and tended to be decreased in MSA. MSA and PD\u0026thinsp;+\u0026thinsp;OH were highly efficiently separated when individual values for CSF/plasma ratios of DHPG were expressed as a function of cardiac \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity (blue and pink rectangles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and when cardiac arteriovenous increments in plasma DHPG were expressed as a function of cardiac NE spillovers (blue and pink rectangles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThere is a longstanding need for pathophysiologically relevant biomarkers that can separate MSA from PD\u0026thinsp;+\u0026thinsp;OH. In the present retrospective analysis we obtained evidence that CSF/plasma ratios of DHPG, CSF/plasma ratios of NE, cardiac arteriovenous increments in plasma DHPG, cardiac NE spillovers, and cardiac \u003csup\u003e18\u003c/sup\u003eF-DA-derived radioactivity all distinguish MSA from PD\u0026thinsp;+\u0026thinsp;OH. The following discussion explains why these findings make sense from a pathophysiological point of view.\u003c/p\u003e \u003cp\u003eCSF DHPG probably mainly reflects central neural stores of NE, which are depleted in both PD\u0026thinsp;+\u0026thinsp;OH and MSA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the periphery, however, there is sympathetic noradrenergic deficiency in PD\u0026thinsp;+\u0026thinsp;OH [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], whereas in MSA sympathetic noradrenergic innervation and function generally are intact [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Accordingly, one would expect that CSF/plasma ratios of DHPG and NE would be lower in MSA than in PD\u0026thinsp;+\u0026thinsp;OH. When CSF/plasma ratios of DHPG and NE were considered together, none of the MSA patients had values for both variables within the control range, whereas most of the PD\u0026thinsp;+\u0026thinsp;OH patients did. These data suggest that the finding of low CSF/plasma ratios of DHPG and NE offers a positive biomarker for MSA compared to PD\u0026thinsp;+\u0026thinsp;OH. The relative preservation of peripheral DHPG levels in MSA despite central NE depletion likely reflects intact peripheral sympathetic innervation and normal intra-neuronal enzymatic metabolism of cytoplasmic NE, in contrast with denervation and a shift from vesicular sequestration to enzymatic metabolism in PD\u0026thinsp;+\u0026thinsp;OH [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe sympathetic noradrenergic lesion in PD\u0026thinsp;+\u0026thinsp;OH is especially prominent in the heart [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, reported previously that patients with PD\u0026thinsp;+\u0026thinsp;OH have markedly decreased cardiac NE spillovers even when total body NE spillovers are within normal limits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We have also reported previously that PD\u0026thinsp;+\u0026thinsp;OH entails attenuated cardiac arteriovenous increments in plasma DHPG levels [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this study we found that combining cardiac NE spillovers with cardiac arteriovenous increments in plasma DHPG levels completely separated MSA from PD\u0026thinsp;+\u0026thinsp;OH, whereas arm arteriovenous increments in plasma DHPG were ineffective in this regard.\u003c/p\u003e \u003cp\u003eThe present neurochemical and sympathetic neuroimaging findings add to growing evidence from cross-sectional studies that one can separate groups of patients with MSA or PD\u0026thinsp;+\u0026thinsp;OH by biomarkers. Other biomarkers include measures of olfactory dysfunction [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], diffusion tensor magnetic resonance imaging [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], a-syn seeding activity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], a-syn deposition in skin biopsies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and CSF neurofilament light chain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The relative efficiencies of these and other modalities, alone and especially in combination, for distinguishing MSA from PD\u0026thinsp;+\u0026thinsp;OH remain to be assessed.\u003c/p\u003e \u003cp\u003eOverall, our data provide new support for DHPG measurements as a practical and potentially scalable clinical test for distinguishing MSA from PD\u0026thinsp;+\u0026thinsp;OH. Even without cardiac catheterization, the CSF/plasma DHPG ratio alone yields respectable separation between MSA and PD\u0026thinsp;+\u0026thinsp;OH in ROC analyses, suggesting potential utility in academic autonomic medicine centers.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThe participants in this study were highly selected and studied at a single site, and they agreed to undergo comprehensive testing without the presumption of personal benefit. Generalizability to the overall population of LBD patients is unknown.\u003c/p\u003e \u003cp\u003eRelatively few groups in the United States carry out right heart catheterizations and arterial blood sampling, and none measure cardiac NE spillover, which requires both infusion of tracer-labeled NE and means to quantify coronary sinus blood flow. The present study suggests that cardiac arteriovenous increments in plasma DHPG may be sufficient to separate MSA from PD\u0026thinsp;+\u0026thinsp;OH in individual patients. This seems within the capability of cardiology investigators who evaluate patients with neurocardiological disorders at academic centers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCommercial laboratories do not currently offer DHPG assays, and liquid chromatography with electrochemical detection, which has been a standard method used for many years [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], is becoming impractical because of the lack of availability of electrodes suitable for series electrochemical detection. Perhaps the present results will help convince laboratories that assay catecholamines by liquid chromatography with tandem mass spectroscopy to add DHPG to their repertoires.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImplications\u003c/h2\u003e \u003cp\u003eThis report introduces neurochemical biomarkers that separate PD\u0026thinsp;+\u0026thinsp;OH from MSA based on CSF and plasma DHPG levels. Prospective studies are needed to determine whether these biomarkers predict the trajectory of phenoconversion from clinically defined pure autonomic failure to central synucleinopathies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Neurochemical biomarkers assessed in combination with other modalities such as skin biopsies with immunofluorescence confocal microscopy [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] or a-syn seed amplification assays [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] may lead to a multimodal framework that would enable diagnosis by convergence.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research reported here was supported (in part) by the research Division of Intramural Research, National Institutes of Health (NINDS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support:\u0026nbsp;\u003c/strong\u003eDivision of Intramural Research, NINDS, NIH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST:\u003c/strong\u003e The authors have no conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSG:\u0026nbsp;\u003c/strong\u003eStudy concept, data analysis, manuscript writing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePS:\u0026nbsp;\u003c/strong\u003eData acquisition, data analysis, manuscript editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCH:\u0026nbsp;\u003c/strong\u003eData acquisition, data analysis, manuscript editing\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdo WF, Bloem BR, Van Geel WJ, Esselink RA, Verbeek MM (2007) CSF neurofilament light chain and tau differentiate multiple system atrophy from Parkinson\u0026apos;s disease. 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Brain 117:835-845\u003c/li\u003e\n\u003cli\u003eYuan Y, Wang Y, Liu M, Luo H, Liu X, Li L, Mao C, Yang T, Li S, Zhang X, Gao Y, Xu Y, Yang J (2024) Peripheral cutaneous synucleinopathy characteristics in genetic Parkinson\u0026apos;s disease. Front Neurol 15:1404492\u003c/li\u003e\n\u003cli\u003eZiegler MG, Lake CR, Kopin IJ (1977) The sympathetic-nervous-system defect in primary orthostatic hypotension. N. Engl. J. Med. 296:293-297\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-autonomic-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"autr","sideBox":"Learn more about [Clinical Autonomic Research](http://link.springer.com/journal/10286)","snPcode":"10286","submissionUrl":"https://www.editorialmanager.com/autr/default2.aspx","title":"Clinical Autonomic Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DHPG, Parkinson, multiple system atrophy, orthostatic hypotension, sympathetic","lastPublishedDoi":"10.21203/rs.3.rs-6675849/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6675849/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe autonomic synucleinopathy multiple system atrophy (MSA) can be difficult to distinguish clinically from Parkinson disease with orthostatic hypotension (PD\u0026thinsp;+\u0026thinsp;OH). \u003csup\u003e18\u003c/sup\u003eF-Dopamine positron emission tomography separates these conditions based on cardiac noradrenergic deficiency in PD\u0026thinsp;+\u0026thinsp;OH and not in MSA but is available only at the NIH Clinical Center. 3,4-Dihydroxyphenylglycol (DHPG) is the main neuronal metabolite of norepinephrine. This retrospective observational study examined whether DHPG levels in cerebrospinal fluid (CSF) or plasma differentiate MSA from PD\u0026thinsp;+\u0026thinsp;OH.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe reviewed CSF and plasma neurochemical data from all patients referred for evaluation at the NIH Clinical Center between 1995 and 2024 for chronic autonomic failure or parkinsonism. A concurrently studied comparison group were healthy volunteers or patients with orthostatic intolerance.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCSF DHPG was decreased in MSA (N\u0026thinsp;=\u0026thinsp;46, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to the controls but also tended to be decreased in PD\u0026thinsp;+\u0026thinsp;OH (N\u0026thinsp;=\u0026thinsp;16, p\u0026thinsp;=\u0026thinsp;0.0598). Antecubital venous plasma DHPG was decreased in PD\u0026thinsp;+\u0026thinsp;OH (N\u0026thinsp;=\u0026thinsp;40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but also in MSA (N\u0026thinsp;=\u0026thinsp;59, p\u0026thinsp;=\u0026thinsp;0.0458). CSF/plasma concentration ratios of DHPG were lower in MSA than in PD\u0026thinsp;+\u0026thinsp;OH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Cardiac arteriovenous increments in plasma DHPG and cardiac norepinephrine spillovers were strikingly decreased in PD\u0026thinsp;+\u0026thinsp;OH (N\u0026thinsp;=\u0026thinsp;6) and were lower than in MSA (N\u0026thinsp;=\u0026thinsp;20, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 each). Combining cardiac arteriovenous increments in plasma DHPG with norepinephrine spillovers completely separated PD\u0026thinsp;+\u0026thinsp;OH from MSA.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCSF/plasma ratios of DHPG, cardiac venous-arterial differences in plasma DHPG, and cardiac norepinephrine spillovers separate MSA from PD\u0026thinsp;+\u0026thinsp;OH. From our results we propose that biomarker combinations involving DHPG in biofluids may enable a pathophysiological differential diagnosis of MSA vs. PD\u0026thinsp;+\u0026thinsp;OH.\u003c/p\u003e","manuscriptTitle":"3,4-Dihydroxyphenylglycol levels separate multiple system atrophy from Parkinson disease with orthostatic hypotension","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 10:04:08","doi":"10.21203/rs.3.rs-6675849/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-26T15:29:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-19T17:53:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-18T06:45:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Autonomic Research","date":"2025-05-15T19:13:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-autonomic-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"autr","sideBox":"Learn more about [Clinical Autonomic Research](http://link.springer.com/journal/10286)","snPcode":"10286","submissionUrl":"https://www.editorialmanager.com/autr/default2.aspx","title":"Clinical Autonomic Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"675559b0-7bfe-43fa-b3ac-4e69074d076b","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T16:00:34+00:00","versionOfRecord":{"articleIdentity":"rs-6675849","link":"https://doi.org/10.1007/s10286-025-01150-8","journal":{"identity":"clinical-autonomic-research","isVorOnly":false,"title":"Clinical Autonomic Research"},"publishedOn":"2025-09-17 15:57:11","publishedOnDateReadable":"September 17th, 2025"},"versionCreatedAt":"2025-05-22 10:04:08","video":"","vorDoi":"10.1007/s10286-025-01150-8","vorDoiUrl":"https://doi.org/10.1007/s10286-025-01150-8","workflowStages":[]},"version":"v1","identity":"rs-6675849","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6675849","identity":"rs-6675849","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}