8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease.

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

Carbonic anhydrase II in the cerebrospinal fluid: its value as a disease marker.

Carbonic anhydrase (CA) II is the predominant CA isoenzyme in the brain of mammals. We have recently developed a dual-label time-resolved immunofluorometric assay to quantify minute amounts of CA I and II in the cerebrospinal fluid (CSF). The present study was aimed at elucidating the clinical value of such measurements in the case of neurological disorders. Lumbar CSF samples were obtained from 111 patients suffering from various neurological diseases and from 97 control patients with no specific signs of central nervous system diseases. The highest CA II concentrations were found in patients with brain infarction (median 66.5 micrograms L-1, n = 20), whereas the control patients had markedly lower values (median 7.8 micrograms L-1, n = 97). Relative to a reference range calculated from the control material (10.2 +/- 17.2 micrograms L-1), the sensitivity of CA II measurement in differentiating brain infarction was 100%. Patients with transient ischaemic attack (median 11.2 micrograms L-1, n = 9), multiple sclerosis (median 14.7 micrograms L-1, n = 18) or epilepsy (median 20.3 micrograms L-1, n = 17) usually had CA II concentrations within the normal range, but those with central nervous system infection (n = 14), dementia (n = 19) or trigeminal neuralgia (n = 6) tended to have higher CA II levels in their CSF, the median values being 39.1 micrograms L-1, 45.5 micrograms L-1 and 44.0 micrograms L-1 respectively. The findings indicate that the concentration of CA II in the CSF marks disease activity in patients with brain damage. This finding could provide a basis for further studies estimating the value of CA II measurement as a new laboratory marker of diseases affecting the brain.

A competitive dual-label time-resolved immunofluorometric assay for simultaneous detection of carbonic anhydrase I and II in cerebrospinal fluid.

Carbonic anhydrase (CA) is functionally an important enzyme in the central nervous system (CNS) where it is involved in the control of acid-base balance and regulation of the production of cerebrospinal fluid (CSF). Isoenzyme II (CAII) is the most widely distributed CA in the CNS being specifically present in CNS glial tissue and therefore it is expected to be leaked to CSF in degenerative CNS diseases. A competitive dual-labeled time-resolved immunofluorometric assay was developed for simultaneous quantification of human CAI (HCA I) and II (HCA II) in CSF. HCA I was measured to determine the blood contamination in the samples. This solid-phase immunoassay is based on competition between europium (Eu3+)- or samarium (Sm3+)-labeled antigen and the sample antigens for polyclonal rabbit antibodies which are attached to microtiter-plate wells precoated with sheep anti-rabbit IgG. The subsequent immunoassay, including the separation of free and bound HCA I and II, requires only one incubation step, after which an enhancement solution dissociates Sm3+ and Eu3+ ions from the labeled HCA I and II, respectively, into a solution where they form highly fluorescent chelates. Spectra of the fluorescent chelates in the microtitration strip wells were run on time-resolved fluorometers equipped with filters for Eu3+ (613 nm) and Sm3+ (643 nm), the fluorescence from each sample being inversely proportional to the concentration of antigens. The detection limit of the HCA II assay was 0.3 micrograms/l and that of the HCA I assay was 5.2 micrograms/l. The intra- and inter-assay imprecisions (C.V.s) were 8.0% and 8.8% for HCA I and 6.3% and 4.8% for HCA II, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A single-step solid phase radioimmunoassay for quantifying human carbonic anhydrase I and II in cerebrospinal fluid.

A single-step solid phase radioimmunoassay was developed to detect human carbonic anhydrase (CA) isoenzymes I (CA I) and II (CA II) in cerebrospinal fluid (CSF). The assay is capable of routinely detecting both isoenzymes at ng levels compared to the microgram levels of the traditional catalytic methods, which failed to demonstrate any CA activity in CSF. When the values of immunoreactive CA II in CSF were corrected for blood contamination (the CA I/CA II ratio of blood was about 7.9), the amount of brain tissue originated CA II could be calculated. The CA II values in CSF samples from 13 patients with multiple sclerosis were higher than those in CSF samples from 11 patients with various peripheral neurological disorders. Since CA II has been specifically localized to oligodendrocytes and myelin, our preliminary results suggest the possibility of CA II leakage from oligodendrocytes and myelin into CSF in demyelinating disease.

Physiology and chemistry of cerebrospinal fluid, aqueous humor and endolymph in Squalus acanthias.

By means of the appropriate isotopes injected into the spiny dogfish, Squalus acanthias, the transfer of all major ions into cerebrospinal fluid (CSF), aqueous humor (A) and endolymph (E) was studied. In addition, the effect of raising pCO2 in sea-water upon HCO3- concentration of these fluids was measured. In the several types of experiments, acetazolamide or methazolamide was used to inhibit completely carbonic anhydrase. The rates of fluid formation and ion transfer in CSF and A were fairly close, but those for E were far slower. The general pattern of ion transport in the three fluids were the same, Na+ (or Na+ + K+ in E) entry greater than Cl - entry, and the difference was HCO3-. The greater rate constants for HCO3-, increase in its entry rate by elevation of pCO2, and inhibition of its appearance by the sulfonamides, show that this is a special case of transport; the ion is formed in secretory cells from gaseous CO2 + OH-. Secretory cells at sites of formation of all the fluids contain both carbonic anhydrase and Na+-K+-ATP-ase, which subserve HCO3- formation and Na+ (or K+) transport. Comparison of these results with studies in mammals show that the vertebrate pattern for secretion of these three fluids is well established in the elasmobranch.