Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.     

Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions

Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4–5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.     

Factors influencing myeloid stem cell (CFU-C) survival after cryopreservation of human marrow and chronic granulocytic leukemia cells

Human marrow stem cells obtained from 20 patients (9 with nonhematological malignancy, 11 with acute leukemia in remission) and peripheral blood stem cells from 27 patients with chronic granulocytic leukemia were cryopreserved in 10% dimethyl sulfoxide (Me₂SO). It was found that the optimal cooling rate for the human myeloid stem cells (CFU-C) ranged from 1 to 3 °C per minute. The myeloid stem cells (CFU-C) maintained their viability for up to one year of storage in liquid nitrogen, after an initial 20% reduction due to the freezing procedure. Myeldoid stem cells survived better when thawed and diluted at room temperature (RT) than at 4 °C. However, the viability of thawed stem cells decreased when stored at RT for more than 1 hr. The viability of stem cells cryopreserved in bags and ampoules was similar. No differences were noted in the surivial of normal human marrow stem cells and cells from patients with chronic granulocytic leukemia when cryopreserved under similar conditions.     

Effects of Baseline CSF α-Synuclein on Regional Brain Atrophy Rates in Healthy Elders,Mild Cognitive Impairment and Alzheimer’s Disease

Background

Cerebrospinal fluid (CSF) α-synuclein is reduced in synucleinopathies, including dementia with Lewy bodies, and some studies have found increased CSF α-synuclein in Alzheimer’s disease (AD). No study has explored effects of CSF α-synuclein on brain atrophy. Here we tested if baseline CSF α-synuclein affects brain atrophy rates and if these effects vary across brain regions, and across the cognitive spectrum from healthy elders (NL), to patients with mild cognitive impairment (MCI) and AD.

Methods

Baseline CSF α-synuclein measurements and longitudinal structural brain magnetic resonance imaging was performed in 74 NL, 118 MCI patients and 55 AD patients. Effects of baseline CSF α-synuclein on regional atrophy rates were tested in 1) four pre-hoc defined regions possibly associated with Lewy body and/or AD pathology (amygdala, caudate, hippocampus, brainstem), and 2) all available regions of interest. Differences across diagnoses were tested by assessing the interaction of CSF α-synuclein and diagnosis (testing NL versus MCI, and NL versus AD).

Results

The effects of CSF α-synuclein on longitudinal atrophy rates were not significant after correction for multiple comparisons. There were tendencies for effects in AD in caudate (higher atrophy rates in subjects with higher CSF α-synuclein, P=0.046) and brainstem (higher atrophy rates in subjects with lower CSF α-synuclein, P=0.063). CSF α-synuclein had significantly different effects on atrophy rates in NL and AD in brainstem (P=0.037) and caudate (P=0.006). Discussion: With the possible exception of caudate and brainstem, the overall weak effects of CSF α-synuclein on atrophy rates in NL, MCI and AD argues against CSF α-synuclein as a biomarker related to longitudinal brain atrophy in these diagnostic groups. Any effects of CSF α-synuclein may be attenuated by possible simultaneous occurrence of AD-related neuronal injury and concomitant Lewy body pathology, which may elevate and reduce CSF α-synuclein levels, respectively.     

Impact of Cerebrospinal Fluid Shunting for Idiopathic Normal Pressure Hydrocephalus on the Amyloid Cascade

The aim of this study was to determine whether the improvement of cerebrospinal fluid (CSF) flow dynamics by CSF shunting, can suppress the oligomerization of amyloid β-peptide (Aβ), by measuring the levels of Alzheimer’s disease (AD)-related proteins in the CSF before and after lumboperitoneal shunting. Lumbar CSF from 32 patients with idiopathic normal pressure hydrocephalus (iNPH) (samples were obtained before and 1 year after shunting), 15 patients with AD, and 12 normal controls was analyzed for AD-related proteins and APLP1-derived Aβ-like peptides (APL1β) (a surrogate marker for Aβ). We found that before shunting, individuals with iNPH had significantly lower levels of soluble amyloid precursor proteins (sAPP) and Aβ38 compared to patients with AD and normal controls. We divided the patients with iNPH into patients with favorable (improvement ≥ 1 on the modified Rankin Scale) and unfavorable (no improvement on the modified Rankin Scale) outcomes. Compared to the unfavorable outcome group, the favorable outcome group showed significant increases in Aβ38, 40, 42, and phosphorylated-tau levels after shunting. In contrast, there were no significant changes in the levels of APL1β25, 27, and 28 after shunting. After shunting, we observed positive correlations between sAPPα and sAPPβ, Aβ38 and 42, and APL1β25 and 28, with shifts from sAPPβ to sAPPα, from APL1β28 to 25, and from Aβ42 to 38 in all patients with iNPH. Our results suggest that Aβ production remained unchanged by the shunt procedure because the levels of sAPP and APL1β were unchanged. Moreover, the shift of Aβ from oligomer to monomer due to the shift of Aβ42 (easy to aggregate) to Aβ38 (difficult to aggregate), and the improvement of interstitial-fluid flow, could lead to increased Aβ levels in the CSF. Our findings suggest that the shunting procedure can delay intracerebral deposition of Aβ in patients with iNPH.     

Serotonin Impairment in CSF of PD Patients,without an Apparent Clinical Counterpart

In Parkinson''s disease (PD), several studies have detected an impaired serotonin (5-HT) pathway, likely affecting both motor and non-motor domains. However, the precise impact of 5-HT impairment is far from established. Here, we have used a HPLC chromatographic method, in a homogenous cohort (n = 35) of non fluctuating, non dyskinetic PD patients, to assess the concentration of 5-HT and its metabolite 5-HIAA in peripheral cerebrospinal fluid (CSF) obtained from lumbar puncture (LP). LP was performed following three days of therapy withdrawal, in order to vanish the effects of prolonged released dopamine agonists (DA), and in absence of any serotonergic agent. The PD patient group showed a significantly reduced CSF level of both 5-HT and 5-HIAA compared to either age-matched control subjects (n = 18), or Alzheimer''s disease patients (n = 20). However, no correlation emerged between 5-HT/5-HIAA concentrations and UPDRS-III (r = −0.12), disease duration (r = −0.1), age (r = −0.27) and MMSE (r = 0.11). Intriguingly, low CSF 5-HT levels did not differ for gender or for motor phenotype (in terms of non-tremor dominant subtype and tremor dominant subtype). Further, low CSF 5-HT levels did not correlate with the presence of depression, apathy or sleep disturbance. Our findings support the contention that 5-HT impairment is a cardinal feature of stable PD, probably representing a hallmark of diffuse Lewy bodies deposition in the brainstem. However, clinical relevance remains uncertain. Given these findings, an add-on therapy with serotonergic agents seems questionable in PD patients, or should be individually tailored, unless severe depression is present.     

Cerebrospinal fluid homocarnosine in Huntington's disease

The concentration of homocarnosine (γ-aminobutyryl-L-histidine) in the cerebrospinal fluid (CSF) of ten patients with Huntington's disease (HD) and 24 control subjects was determined by high-performance cation exchange chromatography. The mean CSF homocarnosine level was significantly lower in HD patients (0.86 ± 0.16 nmo1/m1) than in controls (1.69 ± 0.18 nmo1/m1).     

Effect of isoniazid on cerebrospinal fluid and plasma GABA levels in Huntington's disease

During a double-blind placebo controlled trial, γ-aminobutyric acid (GABA) was measured in cerebrospinal fluid (CSF) and plasma obtained from patients with Huntington's Disease prior to the start of the trial, at the end of the placebo period and following treatment with isoniazid. The results showed that the GABA concentrations in CSF tripled following treatment with isoniazid although no significant change occurred in plasma GABA levels. This finding in humans indirectly confirms reports of a similar increase of brain GABA content in experimental animals following isoniazid treatment and provides additional evidence that CSF GABA measurements reflect brain GABA activity.     

Heterogeneity of Regional Brain Atrophy Patterns Associated with Distinct Progression Rates in Alzheimer’s Disease

We aimed to identify and characterize subtypes of Alzheimer’s disease (AD) exhibiting different patterns of regional brain atrophy on MRI using age- and gender-specific norms of regional brain volumes. AD subjects included in the Alzheimer''s Disease Neuroimaging Initiative study were classified into subtypes based on standardized values (Z-scores) of hippocampal and regional cortical volumes on MRI with reference to age- and gender-specific norms obtained from 222 cognitively normal (CN) subjects. Baseline and longitudinal changes of clinical characteristics over 2 years were compared across subtypes. Whole-brain-level gray matter (GM) atrophy pattern using voxel-based morphometry (VBM) and cerebrospinal fluid (CSF) biomarkers of the subtypes were also investigated. Of 163 AD subjects, 58.9% were classified as the “both impaired” subtype with the typical hippocampal and cortical atrophy pattern, whereas 41.1% were classified as the subtypes with atypical atrophy patterns: “hippocampal atrophy only” (19.0%), “cortical atrophy only” (11.7%), and “both spared” (10.4%). Voxel-based morphometric analysis demonstrated whole-brain-level differences in overall GM atrophy across the subtypes. These subtypes showed different progression rates over 2 years; and all subtypes had significantly lower CSF amyloid-β_1–42 levels compared to CN. In conclusion, we identified four AD subtypes exhibiting heterogeneous atrophy patterns on MRI with different progression rates after controlling the effects of aging and gender on atrophy with normative information. CSF biomarker analysis suggests the presence of Aβ neuropathology irrespective of subtypes. Such heterogeneity of MRI-based neuronal injury biomarker and related heterogeneous progression patterns should be considered in clinical trials and practice with AD patients.     

7α-hydroxy-3-oxo-4-cholestenoic acid in cerebrospinal fluid reflects the integrity of the blood-brain barrier

There is a continuous flux of the oxysterol 27-hydroxycholesterol (27-OHC) from the circulation across the blood-brain barrier (BBB) into the brain. The major metabolite of 27-OHC in the brain is 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). We confirm a recent report describing the presence of this metabolite in cerebrospinal fluid (CSF) at a relatively high concentration. A simple and accurate method was developed for assay of 7-HOCA in CSF based on isotope dilution-mass spectrometry and use of ²H₄-labeled internal standard. The concentration of this metabolite was found to be markedly increased in CSF from patients with a dysfunctional BBB. There was a high correlation between the levels of 7-HOCA in CSF and the CSF/serum albumin ratio. The concentration of 7-HOCA in CSF was not significantly affected by neurodegeneration. Our findings suggest that 7-HOCA could be used as a diagnostic marker for conditions with a dysfunctional BBB.     

Osmolality of Cerebrospinal Fluid from Patients with Idiopathic Intracranial Hypertension (IIH)

Introduction

Idiopathic intracranial hypertension (IIH) is a disorder of increased intracranial fluid pressure (ICP) of unknown etiology. This study aims to investigate osmolality of cerebrospinal fluid (CSF) from patients with IIH.

Methods

We prospectively collected CSF from individuals referred on suspicion of IIH from 2011–2013. Subjects included as patients fulfilled Friedman and Jacobson’s diagnostic criteria for IIH. Individuals in whom intracranial hypertension was refuted were included as controls. Lumbar puncture with ICP measurement was performed at inclusion and repeated for patients after three months of treatment. Osmolality was measured with a Vapor Pressure Osmometer.

Results

We collected 90 CSF samples from 38 newly diagnosed patients and 28 controls. At baseline 27 IIH-samples and at 3 months follow-up 35 IIH-samples were collected from patients. We found no significant differences in osmolality between 1) patients at baseline and controls (p = 0. 86), 2) patients at baseline and after 3 months treatment (p = 0.97), and 3) patients with normalized pressure after 3 months and their baseline values (p = 0.79). Osmolality in individuals with normal ICP from 6–25 cmH₂O (n = 41) did not differ significantly from patients with moderately elevated ICP from 26–45 cmH₂O (n = 21) (p = 0.86) and patients with high ICP from 46–70 cmH₂O (n = 4) (p = 0.32), respectively. There was no correlation between osmolality and ICP, BMI, age and body height, respectively. Mean CSF osmolality was 270 mmol/kg (± 1 SE, 95% confidence interval 267–272) for both patients and controls.

Conclusions

CSF osmolality was normal in patients with IIH, and there was no relation to treatment, ICP, BMI, age and body height. Mean CSF osmolality was 270 mmol/kg and constitutes a reference for future studies. Changes in CSF osmolality are not responsible for development of IIH. Other underlying pathophysiological mechanisms must be searched.     

CSF Biomarkers and Neuropsychological Profiles in Patients with Cerebral Small-Vessel Disease

Despite existing criteria, differential diagnosis of Vascular Dementia (VD) and Alzheimer''s disease (AD) remains difficult. The aim of this study is to figure out cognitive and biomarker profiles that may help to distinguish between VD, AD and AD + Cerebral Small Vessel Disease (CSVD). We examined a cohort of patients with CSVD (n = 92). After stratification of cognitive impaired patients (n = 59) using the standard CSF beta-amyloid 42/40 ratio cut-off point of 0.975, we obtained two groups which differed with respect to several features: 32 patients with normal beta-amyloid 42/40 ratio (>0.975) showed markedly impaired blood-brain-barrier function as indicated by an elevated albumin ratio (median 8.35). They also differed in cognitive profiles when compared to 27 patients with AD typical beta-amyloid ratio and normal albumin ratio. We also enrolled an additional group of patients with AD (no significant CSVD on MRI, n = 27) which showed no impairment of the blood-brain-barrier. We showed a negative correlation between the albumin ratio and executive cognitive function (p = 0.016) and a negative correlation between memory function and typical AD markers like Tau (p = 0.004) and p181-Tau (p = 0.023) in our cohort. We suppose that the group of patients with normal beta-amyloid ratio represents VD while patients in the other groups represent AD+CSVD and pure AD. Our results support the idea that a dysfunction of the blood-brain-barrier might be contributing factor in the development of cognitive decline in CSVD as it seems to be of more importance than the severity of white matter lesions.     

Penetration of DGAVP (Org 5667) across the blood-brain barrier in human subjects

To assess the penetration of desglycinamide-arginine-vasopressin (DGAVP, Org 5667) to the central nervous system, levels of DGAVP were measured in the lumbar CSF after peripheral administration. DGAVP (2 mg) was administered intranasally to 37 patients and CSF samples were collected from these patients 5 to 240 minutes later. Detectable levels of DGAVP in CSF could be found 5 minutes after administration, but levels declined rapidly during the next 90 minutes. The DGAVP levels in CSF correlated with plasma levels of DGAVP (r=0.586, p less than 0.001). According to these results, DGAVP may gain access to the central nervous system and may induce central effects.     

Cloning of early erythroid and mixed myeloid/erythroid human bone marrow progenitor cells: comparison of different sources of burst-promoting activity (BPA)

The ability of conditioned media from the 5637 cell line and human placenta (HPCM) to stimulate the in vitro growth of human early erythroid and mixed myeloid/erythroid clones was tested and compared to phytohemagglutinin-stimulated leukocyte supernatants (PHA-LCM). Both 5637 supernatant and PHA-LCM were equally effective with a linear dose-response relationship. HPCM at various concentrations did not exhibit burst-promoting activity (BPA). Thus, "pluripoietin"-containing media provide a large-scale source of BPA similar in its biological activity to standard sources used for studying human hematopoiesis in vitro.     

Effect of increased gene dosage expression on the α-interferon receptors in Down's syndrome

The gene coding for the α,β-interferon (α,β-IFN) receptor is localized to chromosome 21. Cells from patients with Down's syndrome contain an extra chromosome 21, and thereby an expected 1.5-times increase in the number of genes located to this chromosome and in consequence a 1.5-times increase in cell surface α-IFN receptors. Actual measurements of these by competition binding experiments with human recombinant α-IFN on peripheral blood mononuclear cells (PBMC) from patients with Down's syndrome resulted in a mean of 1.69, which is in accordance to the theoretical 1.50, but slightly overestimated due to the calculation method. The increased gene dosage of the α-IFN receptor was quantitatively verified by Southern blot-hybridizations. Further characterization of α-IFN receptor binding showed insignificant differences in dissociation constants among patients and healthy individuals.     

Postburn serum inhibits in vitro production of colony-stimulating factor by mononuclear peripheral blood cells

The effects of postburn serum (PBS) on the production of colony-stimulating factor (CSF) was evaluated in 13 burned patients by adding PBS to normal peripheral blood mononuclear cells (MNC) and assaying the MNC-conditioned media for CSF content. PBS inhibited CSF production by at least 50%. PBS from non-survivors significantly inhibited CSF production more than PBS from survivors. The addition of lithium chloride restored production of CSF in the presence of day 15 PBS but could not overcome the inhibitory effects of day 1 or day 8 PBS. The nature of the inhibitor(s) is uncertain, but correction of the CSF production defect by lithium chloride later in the course of thermal injury suggests that the defect may be reversible.     

Genetic Loci Associated with Alzheimer’s Disease and Cerebrospinal Fluid Biomarkers in a Finnish Case-Control Cohort

Objectives

To understand the relation between risk genes for Alzheimer’s disease (AD) and their influence on biomarkers for AD, we examined the association of AD in the Finnish cohort with single nucleotide polymorphisms (SNPs) from top AlzGene loci, genome-wide association studies (GWAS), and candidate gene studies; and tested the correlation between these SNPs and AD markers Aβ_1–42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF).

Methods

We tested 25 SNPs for genetic association with clinical AD in our cohort comprised of 890 AD patients and 701-age matched healthy controls using logistic regression. For the correlational study with biomarkers, we tested 36 SNPs in a subset of 222 AD patients with available CSF using mixed models. Statistical analyses were adjusted for age, gender and APOE status. False discovery rate for multiple testing was applied. All participants were from academic hospital and research institutions in Finland.

Results

APOE-ε4, CLU rs11136000, and MS4A4A rs2304933 correlated with significantly decreased Aβ_1–42 (corrected p<0.05). At an uncorrected p<0.05, PPP3R1 rs1868402 and MAPT rs2435211 were related with increased t-tau; while SORL1 rs73595277 and MAPT rs16940758, with increased p-tau. Only TOMM40 rs2075650 showed association with clinical AD after adjusting for APOE-ε4 (p = 0.007), but not after multiple test correction (p>0.05).

Conclusions

We provide evidence that APOE-ε4, CLU and MS4A4A, which have been identified in GWAS to be associated with AD, also significantly reduced CSF Aβ_1–42 in AD. None of the other AlzGene and GWAS loci showed significant effects on CSF tau. The effects of other SNPs on CSF biomarkers and clinical AD diagnosis did not reach statistical significance. Our findings suggest that APOE-ε4, CLU and MS4A4A influence both AD risk and CSF Aβ_1–42.     

Serum-free culture of primitive murine hemopoietic colony-forming cells

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.     

Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media – Vitrification versus Slow Freezing Methods

Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco''s modified Eagle medium Ham’s F-12 (DMEM)and K⁺-modified TiProtec (K⁺TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® A_Queous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm² cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K⁺TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K⁺TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).     

Further Characterization of In Vitro Conditions Appropriate for GABA Determination in Human CSF: Impact of Acid Deproteinization and Freeze/Thaw

Recently established standardized protocols for collection, handling, and storage of CSF for measurement of gamma-aminobutyric acid (GABA) have proven valuable in the characterization of various CNS disorders. In response to two recent reports which may have an impact on certain widely used protocols, we have, using the confirmed ion-exchange/fluorometric procedure, systematically evaluated the effects of deproteinization with various concentrations of sulfosalicylic acid (SSA) ranging from 0 to 10% (100 mg/ml), as well as the effects of freeze/thaw (F/T) on CSF GABA levels. Results of F/T studies documented that levels are stable to freezing and thawing. Acid deproteinization studies revealed the presence of an equilibrium between strictly free GABA, demonstrable only in acid-free CSF, and a very loosely bound form of GABA, fully demonstrable only in CSF deproteinized with concentrations of SSA above 1% (10 mg/ml). The relationship between GABA concentrations in undeproteinized and acid-deproteinized CSF revealed a highly significant (p less than .001) correlation, suggesting that alterations of central GABAergic activity would be reflected by either the level of strictly free GABA or free plus loosely bound GABA. This hypothesis was upheld in studies of patients with Parkinson's disease (PD) and Huntington's disease (HD), two neurologic disorders in which dysfunctions of the GABA system have been implicated. Results indicated that CSF GABA levels are significantly reduced in both PD and HD patients compared with neurologically normal controls, whether the measurement is of free GABA or free plus loosely bound GABA. Thus, we conclude that the level of strictly free GABA is stable to freezing and thawing and can only be accurately determined in nonacidified CSF; however, existing protocols employing deproteinization in 5% SSA yield data that provide an equally good reflection of central GABAergic transmission.