Preparation of a pipette tip-based molecularly imprinted solid-phase microextraction monolith by epitope approach and its application for determination of enkephalins in human cerebrospinal fluid.

In this study, a novel molecularly imprinted polymer (MIP) monolith for highly selective extraction of enkephalins was synthesized and prepared in a micropipette tip using epitope imprinting technique. The synthesized MIPs were characterized by scanning electron microscope (SEM) and infrared spectroscopy. A molecularly imprinted solid-phase microextraction (MISPME) method was developed for extraction of enkephalins in aqueous solutions. The parameters affecting MISPME were optimized. The results indicated that this MIP monolith exhibited specific recognition capability, high enrichment efficiency and excellent reusability for enkephalins. MALDI-TOF MS analysis demonstrated that this MIP monolith can act as a useful tool for highly selective purification and enrichment of enkephalin, a kind of low abundance protein, from high-abundance proteins in human cerebrospinal fluids (CSF). Employed this MIP monolith as solid-phase microextraction column, quantitative assay of enkephalins in human CSF was developed by HPLC-ultraviolet (UV) detection in this work. The detection limits were 0.05-0.08nM. This MISPME/HPLC-UV method was used to quantify Met-enkephalin and Leu-enkephalin levels in the CSF of patients with cancer pain.

Capillary liquid chromatography and tandem mass spectrometry for the quantification of enkephalins in cerebrospinal fluid.

A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.

The antinociceptive effect of Delta9-tetrahydrocannabinol in the arthritic rat.

Our study addressed the hypothesis that spinal release of endogenous opioids underlies Delta9-tetrahydrocannabinol (Delta9-THC)-induced antinociception in Freund's adjuvant-induced arthritic and nonarthritic rats. The paw-pressure test was used to assess the antinociceptive effects of Delta9-THC versus those of morphine, and opioid and cannabinoid receptor-selective antagonists were used to characterize the involved receptors. Cerebrospinal fluid was collected after Delta9-THC injection (i.p.) for the measurement of endogenous opioid peptides. Our results indicate that morphine or Delta9-THC is equally potent and efficacious in both nonarthritic and arthritic rats. Delta9-THC-induced antinociception is attenuated by the kappa opioid receptor antagonist, nor-binaltorphimine, in arthritic rats only. Delta9-THC induces increased immunoreactive dynorphin A (idyn A) levels in nonarthritic rats while decreasing idyn A in arthritic rats. We hypothesize that the elevated idyn A level in arthritic rats contributes to hyperalgesia by interaction with N-methyl-D-aspartate receptors, and that Delta9-THC induces antinociception by decreasing idyn A release.

Opioid receptor ligands in human hepatic encephalopathy.

BACKGROUND/AIMS: Opioid peptides may contribute to some of the manifestations of hepatic encephalopathy. To address the role of the opioid system in the pathogenesis of hepatic encephalopathy, three representative opioid ligands were measured in plasma and cerebrospinal fluid of patients with hepatic encephalopathy. METHODS: Plasma and cerebrospinal fluid were obtained in three groups of patients: group 1: patients with hepatic encephalopathy; group 2: patients with lumbar back pain; group 3: healthy controls. Met-enkephalin, leu-enkephalin and beta-endorphin levels were measured in extracted plasma and cerebrospinal fluid samples by radioimmunoassay. RESULTS: Plasma met-enkephalin levels were 656% (p<0.05) and 301% (p<0.05) and cerebrospinal fluid met-enkephalin levels were 1481% (p<0.01) and 645% (p<0.05) higher when compared to healthy control and pain control patients, respectively. Although plasma and cerebrospinal leu-enkephalin levels were elevated in patients with hepatic encephalopathy, the increases were not statistically significant. Plasma and cerebrospinal beta-endorphin levels were similar in the three study groups. CONCLUSIONS: The results of this study support accumulating data on the role of the delta opioid receptor ligand met-enkephalin in the pathogenesis of hepatic encephalopathy, and provide a rationale for the use of opioid receptor antagonists in the treatment of hepatic encephalopathy.

Role of PACAP in the relationship between cAMP and opioids in hypoxia-induced pial artery vasodilation.

The opioids methionine enkephalin and leucine enkephalin contribute to hypoxic pial artery dilation in the newborn pig, and adenosine 3',5'-cyclic monophosphate (cAMP) analogs have been shown to elevate cerebrospinal fluid (CSF) opioid concentration. The present study was designed to investigate the contribution of cAMP to hypoxic dilation and to determine whether an endogenous activator of adenylate cyclase, pituitary adenylate cyclase-activating peptide (PACAP), could modulate the cAMP-induced release of opioids to contribute to hypoxic pial dilation in piglets equipped with closed cranial windows. An alpha level of P < 0.05 was considered significant in all statistical tests. Moderate and severe hypoxia (PO2 approximately 35 and 25 mmHg, respectively) induced pial artery dilation that was attenuated by the Rp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate (Rp-8-BrcAMPS), a cAMP antagonist (24 +/- 1 and 36 +/- 2% vs. 21 +/- 1 and 30 +/- 1% for moderate hypoxia and 34 +/- 1 and 46 +/- 2% vs. 24 +/- 1 and 32 +/- 1% for severe hypoxia before and after Rp-8-BrcAMPS, respectively). These responses were associated with an increased CSF cAMP (1,046 +/- 25, 1,366 +/- 28, and 1,735 +/- 47 fmol/ml for control, moderate, and severe hypoxia, respectively). Hypoxic pial dilation was also accompanied by an increase in CSF methionine enkephalin (1,101 +/- 62, 3,283 +/- 119, and 3,835 +/- 129 pg/ml for control, moderate, and severe hypoxia, respectively). Hypoxic dilation additionally increased CSF PACAP (1,727 +/- 86, 2,268 +/- 157, and 7,980 +/- 238 pg/ml for control, moderate, and severe hypoxia, respectively). PACAP (10(-8) and 10(-6) M) elicited pial dilation that was associated with increased CSF cAMP and blunted by Rp-8-BrcAMPS. PACAP-induced dilation was also accompanied by increases in the opioid methionine enkephalin (1,059 +/- 23, 1,483 +/- 34, and 2,108 +/- 77 pg/ml for control and 10(-8) and 10(-6) M PACAP, respectively). These data show that cAMP contributes to hypoxic pial artery dilation. Hypoxia increases CSF PACAP, whereas PACAP elevates CSF opioid concentration. These data, therefore, suggest that PACAP modulates cAMP-induced opioid release, thereby contributing to hypoxic pial dilation.

Role of nitric oxide, cyclic nucleotides, and the activation of ATP-sensitive K+ channels in the contribution of adenosine to hypoxia-induced pial artery dilation.

Previously, it had been observed that nitric oxide (NO) contributes to hypoxia-induced pial artery dilation in the newborn pig. Additionally, it was also noted that activation of ATP-sensitive K+ channels (KATP) contribute to cGMP-mediated as well as to hypoxia-induced pial dilation. Although somewhat controversial, adenosine is also thought to contribute to hypoxic cerebrovasodilation. The present study was designed to investigate the role of NO, cyclic nucleotides, and activation of KATP channels in the elicitation of adenosine's vascular response and relate these mechanisms to the contribution of adenosine to hypoxia-induced pial artery dilation. The closed cranial window technique was used to measure pial diameter in newborn pigs. Hypoxia-induced artery dilation was attenuated during moderate (PaO2 approximately 35 mm Hg) and severe hypoxia (PaO2 approximately 25 mm Hg) by the adenosine receptor antagonist 8-phenyltheophylline (8-PT) (10(-5) M) (26 +/- 2 vs. 19 +/- 2 and 34 +/- 2 vs. 22 +/- 2% for moderate and severe hypoxia in the absence vs. presence of 8-PT, respectively). This concentration of 8-PT blocked pial dilation in response to adenosine (8 +/- 2, 16 +/- 2, and 23 +/- 2 vs. 2 +/- 2, 4 +/- 2, and 6 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after 8-PT, respectively). Similar data were also obtained using adenosine deaminase as a probe for the role of adenosine in hypoxic pial dilation. Adenosine-induced dilation was associated with increased CSF cGMP concentration (390 +/- 11 and 811 +/- 119 fmol/ml for control and 10(-4) M adenosine, respectively). The NO synthase inhibitor, L-NNA, and the cGMP antagonist, Rp 8-bromo cGMPs, blunted adenosine-induced pial dilation (8 +/- 1, 14 +/- 1, and 20 +/- 3 vs. 3 +/- 1, 5 +/- 1, and 8 +/- 3% for 10(-8), 10(-6), and 10(-4) M adenosine before and after L-NNA, respectively). Adenosine dilation was also blunted by glibenclamide, a KATP antagonist (9 +/- 2, 14 +/- 3, 21 +/- 4 vs. 4 +/- 1, 8 +/- 2, and 11 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after glibenclamide, respectively). Finally, it was also observed that adenosine-induced dilation was associated with increased CSF cAMP concentration and the cAMP antagonist, Rp 8-bromo cAMPs, blunted adenosine pial dilation. These data show that adenosine contributes to hypoxic pial dilation. These data also show that NO, cGMP, cAMP, and activation of KATP channels all contribute to adenosine induced pial dilation. Finally, these data suggest that adenosine contributes to hypoxia-induced pial artery dilation via cAMP and activation of KATP channels by NO and cGMP.

Relationship between vasopressin and opioids in hypoxia induced pial artery vasodilation.

It has been observed that a vasopressin receptor antagonist attenuates hypoxic hyperemia in fetal sheep, whereas methionine enkephalin (Met) and leucine enkephalin (Leu) contribute to hypoxia-induced pial artery dilation in newborn pigs. This study was designed to investigate the relationship between vasopressin and opioids in hypoxia-induced pial artery dilation in the newborn pig by use of the closed cranial window technique. Hypoxia-induced pial artery dilation was attenuated during moderate [arterial Po2 (PaO2) approximately 35 mmHg] and severe hypoxia (PaO2 approximately 25 mmHg) by the vasopressin receptor antagonist, [beta-mercapto-beta beta-cyclopentamethylenepropionyl, 2-O-Me-Tyr2, Arg8]vasopressin (MeAVP, 5 micrograms/kg i.v.; 29 +/- 1 vs. 14 +/- 2 and 37 +/- 2 vs. 18 +/- 2% for moderate and severe hypoxia in absence vs. presence of MeAVP, respectively, n = 7). Hypoxia-induced dilation was accompanied by increased cerebrospinal fluid (CSF) vasopressin concentration (26 +/- 1 vs. 67 +/- 4 and 26 +/- 1 vs. 99 +/- 4 pg/ml for control vs. moderate and control vs. severe hypoxia, n = 5). Vasopressin increased CSF Met (895 +/- 28, 1,147 +/- 63, 1,327 +/- 48, and 1,600 +/- 75 pg/ml for control and 40, 400, and 4,000 pg/ml vasopressin, respectively, n = 7). CSF Leu concentration was similarly increased by vasopressin. Furthermore, MeAVP attenuated the release of Met during moderate hypoxia (910 +/- 38 and 2,682 +/- 49 vs. 911 +/- 38 and 2,110 +/- 84 pg/ml for control and moderate hypoxia in absence and presence of MeAVP, respectively, n = 5). MeAVP had similar effects on hypoxia-induced Leu release. These data show that vasopressin contributes to hypoxia-induced pial artery dilation and that vasopressin increases CSF Met and Leu concentrations. These data also suggest that elevated CSF vasopressin concentrations that occur during hypoxemia result in opioid release, which subsequently contributes to hypoxic pial artery dilation.

Relationship between opioids and prostaglandins in hypoxia-induced vasodilation of pial arteries in the newborn pig.

Previously, it has been observed that methionine enkephalin and leucine enkephalin contribute to hypoxia-induced pial artery dilation in the newborn pig. It has also been observed that the cyclooxygenase inhibitor indomethacin attenuates hypoxic hyperemia in piglets. The present study was designed to determine the relationship between opioids and prostaglandins in hypoxia-induced pial artery dilation. Newborn pigs equipped with closed cranial windows were used to measure pial artery diameter and collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids and prostaglandins. Hypoxia-induced artery vasodilation was mildly attenuated during moderate hypoxia (PaCO2 approximately 35 mm Hg), while this response was blunted during severe hypoxia (PaO2 approximately 25 mm Hg) by indomethacin, 5 mg/kg iv (23% +/- 1 % vs 18% +/- 1% and 33% +/- 2% vs 21% +/- 2% for moderate and severe hypoxia in the absence and presence of indomethacin, respectively). Hypoxic dilation was accompanied by increased CSF prostaglandin E2 (PGE2) concentration (1260 +/- 37 vs 1734 +/- 67 and 1256 +/- 33 vs 2859 +/- 189 pg/ml for moderate and severe hypoxia, respectively). Similar changes in CSF 6 keto PGF1alpha concentration during hypoxia were also observed. Topical PGE2 (10,100 ng/ml) increased CSF methionine enkephalin (874 +/- 35, 1290 +/- 44, and 1791 +/- 143 pg/ml for control, 10 and 100 ng/ml PGE2 respectively). Similar increases in CSF methionine enkephalin concentration were observed for topical PGI2. Additionally, these two prostaglandins also increased CSF leucine enkephalin concentration. Furthermore, while indomethacin had no effect on the release of CSF methionine enkephalin during moderate hypoxia, it attenuated the release of this opioid during severe hypoxia (786 +/- 27 and 2633 +/- 74 vs 781 +/- 51 and 2467 +/- 52; 926 +/- 15 and 3489 +/- 156 vs 898 +/- 11 and 2314 +/- 124 pg/ml for control and moderate/severe hypoxia before and after indomethacin, respectively). Similar effects of indomethacin on hypoxic release of leucine enkephalin were also observed. These data indicate that prostaglandins contribute to hypoxic pial dilation. Additionally, these data show that prostaglandins release the opioids methionine enkephalin and leucine enkephalin. Finally, these data suggest that elevated prostaglandin concentrations during severe hypoxia release opioids which in turn contribute to hypoxic pial dilation.

Opioids in cerebrospinal fluid in hypotensive newborn pigs.

This study was designed to determine if opioids were detectable in cerebrospinal fluid (CSF) and if these concentrations were altered by hemorrhagic hypotension. This study was further designed to determine the effects of topically administered opioids on pial arteriolar diameter during normotension and hypotension. Closed cranial windows were used to determine pial arteriolar diameter. Periarachnoid cortical and cisterna magna CSF was collected from piglets during normotension and hypotension (systemic arterial pressure decreased from 63 +/- 1 to 33 +/- 1 mm Hg). Opioid profiles were assessed qualitatively by radioreceptor assay, and individual opioids were measured quantitatively by radioimmunoassay. Periarachnoid cortical and cisterna magna CSF methionine enkephalin-, leucine enkephalin-, dynorphin-, and beta-endorphin-like receptor active values all were increased by hypotension. When quantified by radioimmunoassay, periarachnoid cortical CSF values for methionine enkephalin-like immunoreactivity were 1,167 +/- 58 and 2,975 +/- 139 pg/ml for normotension and hypotension, respectively. Periarachnoid cortical CSF radioimmunoassay values for dynorphin-like immunoreactivity were 15 +/- 2 and 28 +/- 2 pg/ml for normotension and hypotension, respectively. When applied topically to the cortical surface, synthetic methionine enkephalin increased pial arteriolar diameter (134 +/- 4, 158 +/- 4, and 163 +/- 4 microns for control, 574 pg/ml [10(-10) M], and 5,740 pg/ml [10(-9) M], respectively). Similarly, topical synthetic leucine enkephalin and dynorphin elicited pial arteriolar dilation. However, beta-endorphin produced arteriolar constriction. Hypotension attenuated methionine and leucine enkephalin-induced dilation and reversed dynorphin-induced dilation to concentration-dependent constriction. beta-Endorphin-induced constriction was not changed by hypotension. Therefore, opioids could contribute to the control of the cerebral circulation during hypotension.

[Opioid peptides in cerebrospinal fluids of epileptic patients].

By means of RIA, the contents of Leu-enkephalin, Met-enkephalin, and Beta-endorphin in CSF of 32 epileptic patients and 24 controls were determined. It was found that the mean Leu-enkephalin content in CSF of the epileptic patient group was significantly higher than that of the control group (P less than 0.01), whereas the mean contents of Met-enkephalin and Beta-endorphin in CSF showed no significant change as compared with those of the control group. The increase of Leu-enkephalin was not related to such factors as type of seizure, age of onset, length of time after the last seizure, taking of antiepileptic drugs, and abnormality in cranial CT manifestation. This suggested that endogenous opioid peptides might take part in the neurochemical mechanism of human epilepsy, and leu-enkephalin could play an important role in the development of epileptic episodes.

Stereoselective effect of morphine on antinociception and endogenous opioid peptide levels in plasma but not cerebrospinal fluid of dogs.

Morphine releases endogenous opioids into the circulation of dogs. To test the stereospecificity of this effect, as well as to determine whether morphine also releases endogenous opioids centrally, which might be involved in its antinociceptive action, the effects of (-)-morphine sulfate (10 mg/kg, sc) or (+)-morphine hydrobromide on antinociception in a dog tail-flick test, on semi-quantified morphine-induced signs of salivation, emesis, defecation and ataxia, and on the plasma and cerebrospinal fluid (CSF) levels of endogenous opioid peptides were studied. Plasma and CSF levels of immunoreactive beta-endorphin (i-BE), met-enkephalin (i-ME), leu-enkephalin (i-LE), and dynorphin (i-DY) were quantified by radioimmunoassay in octadecylsilyl-silica cartridge extracts. Immunoreactive morphine (i-M) levels were measured in unextracted samples. (-)-Morphine treatment significantly increased antinociception, morphine-induced signs, i-M levels in plasma and CSF, and i-BE, i-ME, and i-LE levels in plasma, but not CSF. Levels of i-DY remained constant in plasma and CSF. (+)-Morphine treatment did not alter any of these parameters, indicating that the effects of morphine on nociception, behavioral signs, and plasma endogenous opioids in dogs were stereoselective. It is concluded that morphine does not cause an increase in immunoreactive endogenous opioid peptides in the CSF at the time of its peak antinociceptive effect.

An inhibitor of endopeptidase-24.15 blocks the degradation of intraventricularly administered dynorphins.

Conversion of the octapeptide dynorphin (Dyn) A-(1-8) to Leu5-enkephalin (LE) by endopeptidase EC 3.4.24.15 (EP-24.15) in vivo was examined using the technique of ventriculocisternal perfusion. Peptides were administered intracerebroventricularly in the presence or absence of the EP-24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFPAAF-pAB) via cannulae placed into the lateral ventricle of urethane-anesthetized rats. The concentration of Dyn-like peptides and LE within the CSF was monitored by radioimmunoassay in samples of CSF taken from a second cannula placed in the cisterna magna. In the absence of inhibitor, less than 5% of the Dyn A-(1-8) administered was recovered in CSF. Immunoreactive LE, which is normally not found in CSF, increased rapidly in content following Dyn A-(1-8) infusion, an observation suggesting that the larger peptide is converted to LE. When the inhibitor cFPAAF-pAB was coadministered with Dyn A-(1-8), the concentration of immunoreactive Dyn A-(1-8) after 5 min was 40 times higher than that found in the absence of inhibitor. The angiotensin converting enzyme inhibitor captopril reduced the degradation of Dyn A-(1-8) to a much lesser degree. The inhibitor of EP-24.15 also afforded some protection of other Dyn-like peptides. No EP-24.15 activity was found in rat CSF, whereas high activity was found in the choroid plexus. Taken together, these data clearly indicate that an ectoenzyme form of EP-24.15 rapidly converts intracerebroventricularly administered Dyn-like peptides to LE.

A study on opioid peptides in CSF of patients with epilepsy.

The contents of L-EK, M-EK and beta-EP in CSF of 32 epileptics and 24 controls were determined by RIA. The mean L-EK content of epileptics was significantly higher than that of the controls (P less than 0.01). There were no obvious changes with respect to mean M-EK and beta-EP contents. No significant differences were seen in L-EK contents between generalized and partial seizures, treated and untreated with antiepileptic drugs, normal and abnormal CT manifestation patient groups. These data indicated that endogenous L-EK content was related to human epilepsy, and changes in opioid peptides were selective changes shared by different types of seizures. The increase of L-EK content was not caused by taking antiepileptic drugs, nor due to structural pathological changes of the brain that might be found on CT scanning, but a manifestation of neurochemical disorders of the brain that resulted in epilepsy.

Hemodynamic responses of conscious rats following intrathecal injections of prodynorphin-derived opioids: independence of action of intrathecal arginine vasopressin.

Experiments were conducted (i) to determine the hemodynamic (blood pressure and heart rate) responses of conscious rats following intrathecal (IT) administration of endogenous prodynorphin-derived opioids into the lower thoracic space, (ii) to identify the receptors involved in mediating their cardiovascular responses, and (iii) to reveal any possible hemodynamic interactions with the neuropeptide arginine vasopressin. Male Sprague-Dawley rats were surgically prepared with femoral arterial and venous catheters as well as a spinal catheter (into lower thoracic region, T9-T12). After recovery, hemodynamic responses were observed in conscious rats for 5-10 min after IT injections of artificial cerebrospinal fluid (CSF) solution, prodynorphin-derived opioids (dynorphin A, dynorphin B, dynorphin A (1-13), dynorphin A (1-10), alpha- and beta-neoendorphin, leucine enkephalin (LE), methionine enkephalin (ME), arginine vasopressin (AVP), or norepinephrine (NE)). IT injections of AVP (10 or 20 pmol), dynorphin A (1-13), or dynorphin A (10-20 nmol) caused pressor effects associated with a prolonged and significant bradycardia. Equimolar (20 nmol) concentrations of LE, ME, alpha- and beta-neoendorphin, and dynorphin A (1-10) caused no significant blood pressure or heart rate changes. Combined IT injections of dynorphin A (1-13) and AVP caused apparent additive pressor effects when compared with the same dose of either peptide given alone. IT infusion of the specific AVP-V1 antagonist d(CH2)5Tyr(Me)AVP before subsequent IT AVP, dynorphin A (1-13), or NE administration inhibited only the subsequent pressor responses to AVP. The kappa-opioid antagonist (Mr2266) infused IT blocked the pressor actions of subsequent dynorphin A administration and not AVP or NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid by microbore high-performance liquid chromatography and capillary zone electrophoresis coupled to tandem mass spectrometry.

The performance of microbore high-performance liquid chromatography and capillary zone electrophoresis, both equipped with on-line tandem mass spectrometric detection capability, was evaluated critically for the determination of endogenous amounts of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid. Using an identical sample clean-up and enrichment procedure, capillary zone electrophoresis-mass spectrometry is limited in its concentration detection capacity owing to its much smaller injection volume. Leucine enkephalin was identified in post-mortem equine cerebrospinal fluid at the 1-5 ng/ml level by liquid chromatography-mass spectrometry.

[A study of the leucine enkephalin like substance and enkephalin degrading enzyme activity in human cerebrospinal fluid].

Leucine enkephalin like substance (Leu-Enk) and enkephalin degrading enzyme activity (EDEA) in cerebrospinal fluid (CSF) of three patients suffering from pain were measured. As compared with control values before treatments, the values of Leu-Enk in CSF decreased, and those of EDEA increased in all the patients. After the treatment, two patients were fortunately cured of pain, and their two values returned to normal. But the other patient could not recover from severe pain, and their values remained abnormal. It is concluded that Leu-Enk and enkephalin degrading enzyme in CSF may play an important role in regulation of pain sensitivity.

[An experimental study on the contents of enkephalins in the cerebrospinal fluid in rabbits with unilateral destructive lesions produced in the substantia nigra and their relation to the regulation of MIF-1].

The changes of the contents of enkephalins in cerebrospinal fluid (CSF) and the action of MIF-1 in rabbit experimental models of Parkinson's disease were studied. In the experiment, the rabbits received injection of 6-hydroxydopamine (6-OH DA) into the unilateral substantia nigra. The contents of Met-enkephalin (MEK) and Leuenkephalin (LEK) in the CSF of the fourth ventricles of the normal control rabbits and those with destructive lesions in the substantia nigra were determined with radio-immunoassay. The concentrations of MEK and LEK in CSF of the rabbit models increased markedly to 14.3 and 28.2 folds in the controls respectively. The increased enkephalin content in CSF could be reduced to the normal level by intravenous administration of MIF-I. The results indicated that the action of MIF-I may be one of the important factors in alleviating the symptoms of parkinsonian patients.

[The concentration of homovanillic acid and leucine-enkephalin in the lumbar CSF of patients with Parkinson's disease before and after madopar treatment].

For comparison, the concentrations of homovanillic acid (HVA) and leucine-enkephalin (LEK) in the lumbar cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) before and after treatment with madopar were determined 19 patients, suffering from common surgical diseases, who had CSF withdrawn by lumbar punctures for spinal anesthesia, served as controls. HVA was estimated by fluorescence scanning after polyamide thin-layer chromatography. LEK was measured with radio-immuno-assay. The mean concentration of HVA from 22 patients before madopar treatment (0.085 +/- 0.011 mg/L, means +/- S means) was significantly lower than the control mean value (0.264 +/- 0.022 mg/L) (P less than 0.001); while that of LEK from 25 patients before treatment (197.01 +/- 12.96mg/L, means +/- S means) was significantly higher than the control mean value (88.79 +/- 8.66mg/L) (P less than 0.001). Treatment with madopar increased the concentration of HVA (0.222 +/- 0.032mg/L) (P less than 0.001) but exhibited no significant effect on the levels of LEK. It is confirmed that there is a reduction of HVA in CSF in Parkinsonian patients. The results suggest that an increase of enkephalin in the brain seems to play some role in the pathogenesis of PD, but why madopar could not significantly alter the LEK level in CSF awaits further studies.