Toward a reliable distinction between patients with mild cognitive impairment and Alzheimer-type dementia versus major depression.

BACKGROUND: Cerebrospinal fluid (CSF) levels of soluble amyloid precursor protein (sAPP) and its alpha-secreted form (alpha-sAPP) were investigated as a means to distinguish between individuals with mild cognitive impairment (MCI) and Alzheimer-type dementia (DAT) and those with major depressive episode (MDE) showing secondary memory deficits. METHODS: Twenty-seven patients with MCI, 32 with probable DAT, and 24 with MDE attending a memory clinic were studied. Cerebrospinal fluid levels of sAPP/amyloid precursor-like protein 2 (APLP2) and alpha-sAPP were detected by Western blotting. RESULTS: Patients with MDE had the highest CSF levels of total sAPP/APLP2 as compared with MCI and DAT patients (p < .001); sAPP/APLP2 levels were higher in MCI than in DAT subjects. Whereas alpha-sAPP levels did not differ between the MCI and DAT groups, median levels of this peptide were significantly lower in MCI and DAT versus MDE patients. CONCLUSIONS: Soluble amyloid precursor protein/APLP2 and alpha-sAPP concentrations in CSF can differentiate between DAT and MCI versus MDE, facilitating early ameliorative interventions and appropriate treatment regimens.

Concentrations of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), IGF, and IGFBP-3 protease activity in cerebrospinal fluid of children with leukemia, central nervous system tumor, or meningitis.

Insulin-like growth factor-II (IGF-II) is the major IGF in human cerebrospinal fluid (CSF), whereas IGF-I is only detectable in trace amounts. The major IGFBPs in CSF are IGFBP-2 and IGFBP-4. Normally, IGFBP-3 is a minor component in CSF of healthy subjects, but may be increased in pathological states. We investigated IGF-I, IGF-II, and IGFBP-3 levels by specific RIAs in CSF from patients with central nervous system (CNS) tumor or leukemia and compared them with values in patients with meningitis. Further, as proteolysis of IGFBP-3 is part of the modulation of IGF activity, IGFBP-3 fragmentation was quantified by densitometric analysis of [125I]IGFBP-3 protease assays. We examined CSFs of 23 children with malignant CNS tumors, 18 children with leukemia, and 13 children with meningitis. The CSF from 38 children who received lumbar punctures to exclude meningitis was used to define the normal range for IGF-I, IGF-II, IGFBP-3, and IGFBP-3 protease activity in CSF. CNS tumor and leukemia patients had normal levels of IGF-I and IGF-II in CSF, whereas the IGF-II concentration in CSF of meningitis patients was elevated (P < 0.0001). Only 2 of 13 (15%) meningitis patients had elevation of CSF IGFBP-3 concentrations, despite high numbers of inflammatory cells. By comparison, elevated IGFBP-3 concentrations were found in the CSF of 16 of 23 (70%) CNS tumor patients and 6 of 7 (86%) CNS tumor patients with microscopically detectable malignant cells in CSF. Twelve of 13 (92%) patients with medulloblastoma or ependymoma and all 7 medulloblastoma/ependymoma patients with malignant cells in CSF had elevated IGFBP-3 concentrations. The IGFBP-3 protease activity in CSF was elevated in 15 of 16 (94%) patients with CNS tumors of high grade histological malignancy. Five of 6 patients (83%) with acute leukemia and microscopically detectable malignant cells in CSF at the time of diagnosis showed elevated IGFBP-3 concentrations, with normalization after chemotherapy. Leukemia patients without malignant cells in CSF had normal IGFBP-3 concentrations. We conclude that in CSF of children with highly malignant CNS tumor or CNS leukemia, IGFBP-3 is elevated. This phenomenon could be caused by disruption of the blood-CSF barrier and entry of IGFBP-3 from serum, although this appears unlikely, especially for CNS leukemia. More likely possibilities are 1) local production of IGFBP-3 by CNS tumor tissue and secretion into the CSF, or 2) local production of IGFBP-3 by malignant cells within the CSF.

Acute oral captopril inhibits angiotensin converting enzyme activity in human cerebrospinal fluid.

Angiotensin converting enzyme (ACE) activity in human plasma and cerebrospinal fluid (CSF), was measured by a fluorimetric method, following an acute oral dose (75 mg) of captopril. A decrease of approximately 60% in enzyme activity was observed in CSF, suggesting that the drug penetrates the blood/brain barrier (BBB) in sufficient amounts to inhibit the ACE in CSF. The activity of CSF enkephalinase, an enzyme present in human brain and inhibited in vitro by an elevated concentration of captopril, was, however, unaffected by the acute drug administration. It is proposed that the antihypertensive effect of captopril in humans may be due, at least in part, to the inhibition of ACE contained within brain structures.

In vivo 'enkephalinase' inhibition by acetorphan in human plasma and CSF.

Thiorphan, the potent inhibitor of 'enkephalinase', has shown some analgesic properties in experimental animals and in man. The possibility that the intravenous infusion of acetorphan, a prodrug of thiorphan (26 micrograms/kg per min for 60 min), can inhibit plasma and cerebrospinal fluid (CSF) enkephalinase in man in vivo was investigated. A decrease of approximately 65% in enzyme activity was observed in both plasma and CSF. Acetorphan did not induce any significant variation of plasma angiotensin-converting enzyme activity.

Lysosomal hydrolases in cerebrospinal fluid of multiple sclerosis patients. A follow-up study.

The change in activity of lysosomal hydrolases in the brain tissue of patients with demyelinating disease has been suggested to reflect the demyelination process. In this study we measured neutral proteinase (NP), acid proteinase (AP), and beta-glucuronidase (BG) activities in CSF of 32 patients with multiple sclerosis (MS) (remitting, remitting and relapsing, or chronic progressive course of the disease), 62 controls, and 4 patients with chronic inflammatory disease of central nervous system (ID). Samples from MS patients were taken at different clinical conditions of the disease during the 22-month follow-up. Elevated NP activity was found in patients with relapsing course of MS and also in patients with ID (P less than 0.05). NP activity correlated with the number of leucocytes in CSF of both MS (P less than 0.005, r = 0.50) and control (P less than 0.05, r = 0.21) patients. AP activity decreased in the MS group, especially in patients with remitting or remitting and relapsing courses of the disease (P less than 0.05), but even more in patients with ID (P less than 0.01). During the follow-up the increase in NP activity seemed to be associated with the clinical relapses of MS patients. Other enzymes did not fluctuate with the disease. This study suggests that the change in activity of lysosomal hydrolases is not specific for MS. The increase in NP activity in CSF is associated with clinical relapse of individual MS patients during the follow-up and may indicate immunological activation of the demyelination process in the brain. The large intra- and interindividual variation in enzyme activities in the CSF, however, makes the use of these enzymes difficult for diagnosis of MS and follow-up of MS activity.

Reversed-phase high-performance liquid chromatography combined with tandem mass spectrometry in studies of a substance P-converting enzyme from human cerebrospinal fluid.

The application of reversed-phase HPLC in combination with micro-electrospray mass spectrometry to study a substance P (SP)-hydrolysing endoprotease in human cerebrospinal fluid (hCSF) is reported. The enzyme was partially purified from the hCSF specimens by ion-exchange chromatography and molecular sieving. During the purification procedure the enzyme activity was monitored by measuring the formation of the SP-fragment 1-7 from SP by radioimmunoassay. Regarding its behaviour upon molecular sieve chromatography, the enzyme was suggested to be associated with an apparent molecular mass of around 100 x 10(3). In subsequent experiments using the partially purified endopeptidase, the hydrolysis of SP was demonstrated by HPLC. The reaction product mixture was resolved in several components including the N-terminal fragments 1-8, 1-7 and 1-6 and the C-terminal fragment 8-11. The identity of these fragments were confirmed by tandem mass spectrometry. It was concluded that the present SP-degrading enzyme is different from those previously identified and purified from hCSF. The applied techniques were proven to be highly efficient for the recovery and identification of the released peptide products.

Enkephalinase activity in both physiological and pathological conditions in man.

Among all the enzymes involve din enkephalin degradation "enkephalinase", cleaving the Gly3-Phe4 amide bond, is considered for its specificity with endogenous enkephalins and their receptors. This enzyme, first identified in membrane bound form, has been recently characterized in a soluble one by a new sensitive fluorimetric method substituting the radiometric technique. The possibility to evaluate "enkephalinase" activity in human plasma, amniotic fluid and cerebro spinal fluid (CSF) allows us to investigate its behavior in various physiological and pathological conditions in which alterations of the endogenous opioid system are hypothesized. Our studies were focused on pregnancy, the first period of life, idiopathic headache and opioid addiction. In these conditions "enkephalinase" activity (EKA) generally results increased. In some cases the activity is proportional to the increased amount of substrate, in other cases no correlation seems apparent.

Detection of a soluble form of BACE-1 in human cerebrospinal fluid by a sensitive activity assay.

BACKGROUND: Formation of deposits of the insoluble amyloid beta-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The beta-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. METHODS: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-1. The resulting active enzyme is measured with a chromogenic substrate. The use of 2 coupled reactions produces a detection limit as low as 0.4 pmol/L. RESULTS: The assay detected BACE-1 activity in extracts of human brain tissue as well as, unexpectedly, in human cerebrospinal fluid (CSF). Gel electrophoresis and Western blotting identified the BACE-1 present in CSF as a truncated soluble form of the originally membrane-bound BACE-1. CONCLUSION: Detection of the soluble form of BACE-1 in CSF, a relatively easily accessible biological fluid, may be useful for monitoring the effects of drug candidates in vivo and may have diagnostic or prognostic applications.

CSF BACE1 activity is increased in CJD and Alzheimer disease versus [corrected] other dementias.

To assess the diagnostic utility of CSF BACE1 activity for discriminating Alzheimer disease (AD) from other dementias, particularly Creutzfeldt-Jakob disease (CJD), the authors studied 26 patients with sporadic CJD, 21 patients with AD, and 21 patients with various non-AD, non-CJD dementias (DCs). CSF BACE1 activity was elevated in AD in comparison with DC (p = 0.01). Unexpectedly, CSF BACE1 activity was also increased in sporadic CJD (p = 0.02).

Capillary liquid chromatography-fast atom bombardment mass spectrometry using a high-resolving cation exchanger, based on a continuous chromatographic matrix. Application to studies on neuropeptide peptidases.

Hyphenated mass spectrometric techniques such as LC-MS are advantageous over standard MS methods, because they provide increased sensitivity and minimize signal suppression by other compounds present in the reaction mixture. Recently, we have introduced so-called continuous beds, and applied this technique to prepare a 0.32 mm I.D. cation-exchange capillary column, in order to separate the reaction product substance P(1-7) after proteolytic cleavage of substance P by an endopeptidase recovered from human cerebrospinal fluid. The use of a volatile buffer for elution provides very good flow stability. Ion-exchange microcolumns may be particularly useful for the separation of those peptides that co-elute in reversed-phase chromatography because the separation mechanisms of these two methods are different.

Comparison of X-prolyl dipeptidyl-aminopeptidase activity in human cerebrospinal fluid with that in serum.

X-Prolyl dipeptidyl-aminopeptidase activities in cerebrospinal fluid and serum from the same patients without neurological diseases, undergoing surgery under lumbar anesthesia, were assayed fluorometrically with a newly synthesized fluorogenic substrate, 7-glycylproline-4-methylcoumarinamide; the values were 129.1 +/- 19.5 nmoles/min/l and 54.17 +/- 3.11 mumoles/min/l (mean +/- SEM, n = 23), respectively, and there was no correlation between both activities (r = 0.0894).

Characterization of substance P(1-7) and (1-8) generating enzyme in human cerebrospinal fluid.

A substance P-hydrolyzing endopeptidase has been purified from a large quantity of human cerebrospinal fluid by ion exchange chromatography (DEAE-Sepharose CL-6B) and molecular sieving (Sephadex G-100 and Sephacryl S-200). The purification was monitored by measuring the conversion of synthetic substance P using a radioimmunoassay specific for its (1-7) fragment. The enzyme has an apparent molecular weight of 43,000. It cleaves predominantly at the Phe7-Phe8 and Phe8-Gly9 bonds but gives no or negligible conversion of the other tachykinins, neuromedin K and L (substance K).

[Determination of dipeptidyl aminopeptidase activities in the cerebrospinal fluid]. / Untersuchungen zur Bestimmung von Dipeptidyl-aminopeptidase-Aktivitäten im Liquor cerebrospinalis.

The investigation of enzyme activity in cerebrospinal fluid has been without relevant results for laboratories analysing spinal fluid. For neurochemical purposes, it is interesting that the Substance P is convered by Depeptidyl-aminopeptidase IV (DP IV), liberating dipeptides. The hydrolysis of nitroanilids of the form Xaa-Pro-NHNp in cerebrospinal fluid was analysed using them as peptidases substrates. Finally a method for measuring the activity was proposed.

Alterations in cerebrospinal fluid angiotensin II by sodium intake in patients with essential hypertension.

1. Angiotensin (ANG) levels were measured in the cerebrospinal fluid of 15 patients with essential hypertension on a high sodium diet for 1 week and on a low sodium diet for a further week. ANGs were determined using a system of extraction by Sep-Pak cartridges followed by h.p.l.c. combined with radioimmunoassay. 2. Sodium depletion resulted in increases of ANG II in the cerebrospinal fluid from 1.16 +/- 0.38 (SEM) to 1.83 +/- 0.43 fmol/ml (P less than 0.01) and of ANG III from 0.65 +/- 0.11 to 0.86 +/- 0.15 fmol/ml (P less than 0.01). 3. The ANG II level in the cerebrospinal fluid was found to be unchanged and recovery of added ANG II was approximately 90%, even after incubation for 3 h, on both diets. Thus, it is unlikely that ANG II is produced or degraded in the cerebrospinal fluid in vitro. 4. There was no significant correlation between the cerebrospinal fluid and the plasma ANG II concentration on the low sodium diet. 5. These results suggest that the cerebrospinal fluid ANG II level increases with sodium depletion, and that the effect of the level of ANG II on the activity of the angiotensin-forming system in the central nervous system may be assessed by determination of ANG II in the cerebrospinal fluid in patients with essential hypertension.

Assay of preproenkephalin A processing enzymes in human lumbar cerebrospinal fluid.

Vmax and Km measurements have been obtained for endogenous peptidases that are important for methionine enkephalin (ME) homeostasis in humans. Those peptidases in human lumbar cerebrospinal fluid (CSF) act upon several synthetic biologically significant peptides that are also contained within the preproenkephalin Ahuman,1-267 molecule. The amount of endogenous methionine enkephalin-like immunoreactivity (ME-li) in human lumbar CSF is 74.1 +/- 5.7 fmol ME-li/ml CSF (n = 56; mean +/- SE). The kinetic parameters of the various enzymes that inactivate exogenous, synthetic methionine enkephalin (ME, YGGFM) and that also produce ME from two different portions of the preproenkephalin Ahuman,1-267 precursor molecule were determined. The enzyme that inactivates synthetic ME to FM, and that correlates to the rate of decrease of ME, has a Vmax = 560 +/- 43.3 nmol/ml/min and a Km = 4514 +/- 373 microM (n = 56; mean +/- SE). Preproenkephalin Ahuman,186-193 (PA = YGGFMRGL) was added to CSF samples to characterize those processing and converting enzymes that produce the ME pentapeptide. Vmax, as measured by the rate of the decrease of PA to produce YGGFMR, was 0.192 +/- 0.038 nmol/ml/min and a Km of 513 +/- 121 microM (n = 10; mean +/- SE). Similarly, a bovine analog to preproenkephalin Ahuman,128-140 (PPEhuman, GSEILAKRYGGFM; PPEbovine,125-137, GGEVLGKRYGGFM) was used to characterize that enzyme system that produces ME from an N-terminally extended ME peptide. That endopeptidase had a Vmax of 0.120 +/- 0.048 nmol/ml/min with a Km of 734 +/- 296 microM (n = 10). Those endogenous enzymes in human CSF may relate to the proopiomelanocortin convertase enzymes that contain the subtilisin-like catalytic domain.

Neuropeptide-converting enzymes in cerebrospinal fluid: activities increased in pain from herniated lumbar dis, but not from coxarthrosis.

We measured activities of dynorphin-converting enzyme (DCE), substance P endopeptidase (SPE) and angiotensin-converting enzyme (ACE) in cerebrospinal fluid (CSF) in 13 patients with rhizopathic pain from an herniated lumbar disc, in 9 patients with pain from coxarthrosis and in 11 control patients without pain. In the patients with disc hernia and coxarthrosis, another sample of CSF was analyzed 3-12 months after treatment, when pain had subsided. The DCE activity in the patients was higher than that in both the control patients and the patients with pain from coxarthrosis (nociceptive pain). Similarly, the activity of SPE was lower in the patients with herniated lumbar disc than in controls and in the patients with coxarthrosis. After treatment, the difference in activity compared to controls was lower, but still significant in patients with herniated discs. The ACE activity did not differ from controls in patients with ischialgia, while it was increased in patients with coxarthrosis. This increase also remained after arthroplasty with pain relief. In conclusion, measurements of neuropeptides may be useful for evaluating neuropathic pain.

Neutral protease in cerebrospinal fluid from patients with multiple sclerosis and other neurological diseases.

Neutral protease activity was significantly elevated in the cerebro-spinal fluid of patients with multiple sclerosis (MS) in exacerbation and in the acute phase of acute viral meningoencephalitis (AME) compared with that of MS in remission, amyotrophic lateral sclerosis or psychosomatic disease. Since in each relapse of MS, protease activity was higher in exacerbation than in remission, this activity may be one good marker of disease activity in MS. One hundred micro molar of FOY305, synthetic protease inhibitor, inhibited in vitro increased neutral protease activity in MS in exacerbation, which suggests the possibility of a clinical application of this protease inhibitor for MS.

Possible long-lasting inhibition of converting enzyme by enalapril in human cerebrospinal fluid.

1. Converting enzyme and neutral endopeptidase activities were both measured every 3 h by a fluorimetric method in plasma and cerebrospinal fluid of patients diagnosed as migraineurs with aura after lumbar puncture, which was performed 9 h after an acute oral dose of enalapril or placebo. 2. A reduced converting enzyme activity, as compared with placebo, was observed in patients who were given enalapril. On the other hand, neutral endopeptidase activity detected after enalapril did not differ from that measured after placebo. 3. The results seem to indicate that enalapril penetrates the blood-brain barrier in sufficient amounts to reduce converting enzyme activity. Moreover, neutral endopeptidase was not affected by enalapril. Therefore, those clinical effects of the drug which have been attributed to the involvement of central opioid mechanisms may depend on the inhibition of brain converting enzyme but not the inhibition of brain neutral endopeptidase.

Increased angiotensin-(1-7) in hypophysial-portal plasma of conscious sheep.

Studies were undertaken to characterize angiotensin peptides in hypophysial-portal blood of conscious sheep and to determine whether the median eminence (ME) secretes angiotensin peptides into the hypophysial-portal circulation. Simultaneous measurements of angiotensin peptides in jugular and hypophysial-portal plasma were performed in 6 sheep. Cerebrospinal fluid (CSF) was collected and data for hypophysial-portal plasma were corrected for CSF contamination. Angiotensin peptides were also measured in extracts of sheep ME. In a separate group of 4 sheep, simultaneous measurements of angiotensin peptides in arterial and jugular plasma were performed. Using high performance liquid chromatography-based radioimmunoassays, 8 angiotensin peptides were measured: Ang-(1-7), Ang II, Ang-(1-9), Ang I, Ang-(2-7), Ang III, Ang-(2-9), and Ang-(2-10). Renin, angiotensinogen and prolyl endopeptidase were also measured. No differences in angiotensin peptide levels in arterial and jugular plasma were observed. Angiotensin peptide levels in hypophysial-portal plasma were similar to those in jugular plasma, except for Ang-(1-7), the levels of which were 5-fold higher in hypophysial-portal plasma, and Ang I, for which the levels in hypophysial-portal plasma were 46% of the jugular levels. Renin and angiotensinogen levels were similar in arterial, jugular, and hypophysial-portal plasma. Angiotensin peptide contents of sheep ME were less than 16 fmol/ME. However, the prolyl endopeptidase content of sheep ME was 430-fold higher than plasma levels. The low levels of angiotensin peptides in sheep ME indicate that it does not secrete these peptides into the hypophysial-portal circulation. Rather, the high level of prolyl endopeptidase in ME is consistent with region-specific metabolism of Ang I delivered to the ME by arterial blood, generating increased levels of Ang-(1-7) in hypophysial portal plasma. The increased levels of Ang-(1-7) in hypophysial-portal plasma may play a role in regulation of pituitary function.