Human amyloid-b synthesis and clearance rates as measured in cerebrospinal fluid in vivo Randall J Bateman 1–3 , Ling Y Munsell 4 , John C Morris 1,2,5 , Robert Swarm 6 , Kevin E Yarasheski 4 & David M Holtzman 1–3,7 Certain disease states are characterized by disturbances in production, accumulation or clearance of protein. In Alzheimer disease, accumulation of amyloid-b (Ab) in the brain and disease-causing mutations in amyloid precursor protein or in enzymes that produce Ab indicate dysregulation of production or clearance of Ab. Whether dysregulation of Ab synthesis or clearance causes the most common form of Alzheimer disease (sporadic, 499% of cases), however, is not known. Here, we describe a method to determine the production and clearance rates of proteins within the human central nervous system (CNS). We report the first measurements of the fractional production and clearance rates of Ab in vivo in the human CNS to be 7.6% per hour and 8.3% per hour, respectively. This method may be used to search for novel biomarkers of disease, to assess underlying differences in protein metabolism that contribute to disease and to evaluate treatments in terms of their pharmacodynamic effects on proposed disease- causing pathways. Protein production and clearance are important parameters that are tightly regulated and reflect normal physiology as well as disease states 1–4 . Previous studies of protein metabolism in humans have focused on whole-body or peripheral-body proteins, but not on proteins produced in the CNS. A technique to measure specific protein metabolism in the CNS could provide important insights into CNS protein physiology in health and disease. Certain disease states are characterized by disturbances in protein production, accu- mulation or clearance. In the CNS, disturbances in metabolism of proteins such as the prion protein 5 , alpha-synuclein 6 , tau 7 or Ab 8 can contribute to and, in some cases, cause neurodegenerative diseases such as Creuzfeldt-Jakob disease, Parkinson disease, frontotemporal dementia or Alzheimer disease, respectively. Biochemical, genetic and animal model evidence implicates Ab as a pathogenic peptide in Alzheimer disease. The neuropathologic and neurochemical hallmarks of Alzheimer disease include synaptic loss and selective neuronal death, a decrease in certain neurotransmitters and the presence of abnormal proteinaceous deposits in neurons (neurofibrillary tangles), in the cerebral vasculature (amyloid angiopathy) and in the extracellular space (diffuse and neuritic plaques). The main constituent of plaques is Ab, a peptide of 38–43 amino acids cleaved from the amyloid precursor protein (APP) 9,10 . Throughout life, soluble Ab is secreted mostly by neurons but also other cell types. In late-onset Alzheimer disease, the total amount of Ab that accumulates in brain is B100–200-fold higher in homoge- nates from Alzheimer disease brains than from control brains 11 . Disturbance of Ab production can lead to rare forms of Alzheimer disease in humans. Mutations in three different genes (APP, PSEN1 and PSEN2), which cause early-onset autosomal dominant Alzheimer disease, all result in overproduction of total Ab or Ab 42 (ref. 9). In Down syndrome, three copies of APP result in increased production of Ab, and 100% of individuals with Down syndrome develop Alzheimer disease pathology by age 35 (ref. 12). In late-onset Alzheimer disease (B99% of cases), however, there is not strong evidence for over- production of Ab. Therefore, the underlying cause of deposition of Ab (increased production versus decreased clearance) is not known for most cases of Alzheimer disease. No methods were previously available to quantify protein synthesis or clearance rates in the human CNS. Such a method would be valuable to assess not only Ab synthesis and clearance rates in humans but also the metabolism of a variety other proteins relevant to diseases of the CNS. To address crucial questions about underlying pathogen- esis of Alzheimer disease and Ab metabolism, we developed a method for quantifying the fractional synthesis rate (FSR) and fractional clearance rate (FCR) of Ab in vivo in the human CNS. Our results indicate that by administering a stable isotope-labeled amino acid ( 13 C 6 -leucine), sampling cerebrospinal fluid (CSF) and using high-resolution tandem mass spectrometry to quantify labeled Ab, reproducible rates of Ab synthesis and clearance can be quantified in humans. RESULTS In vivo labeling and quantification of Ab To determine whether labeled Ab (Fig. 1) could be produced and detected in vivo in a human, one individual underwent a 24-h infusion of labeled leucine followed by a lumbar puncture to obtain CSF. We immunoprecipitated Ab from the CSF sample with the Ab-specific Received 2 November 2005; accepted 24 January 2006; published online 25 June 2006; doi:10.1038/nm1438 1 Departments of Neurology, 2 The Alzheimer Disease Research Center, 3 Hope Center for Neurological Disorders 4 Medicine, 5 Pathology and Immunology, 6 Anesthesiology and 7 Molecular Biology & Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8111, St. Louis, Missouri 63110, USA. Correspondence should be addressed to R.J.B. (batemanr@neuro.wustl.edu). 856 VOLUME 12 [ NUMBER 7 [ JULY 2006 NATURE MEDICINE TECHNICAL REPORTS © 2006 Nature Publishing Group http://www.nature.com/naturemedicine