Cerebrospinal fluid levels of immunoreactive substance P and somatostatin in patients with multiple sclerosis and inflammatory CNS disease.

Cerebrospinal fluid (CSF) levels of substance-P like immunoreactivity (SPLI) and somatostatin-like immunoreactivity (SLI) were measured in 43 patients with multiple sclerosis (MS), differentiated according to course and activity of the disease, in 23 patients with inflammatory disease of known bacterial or viral etiology and in 16 control patients using specific radioimmunoassay. SPLI and SLI levels were not significantly different from controls in MS patients whereas SLI was significantly increased in patients with infectious disease of central nervous system and/or subarachnoidal space. It is assumed that CSF SPLI and SLI cannot serve as a diagnostic or prognostic indicator of disease state in multiple sclerosis. Analysis of immunoreactivity by reverse phase HPLC-RIA revealed marked molecular heterogeneity of both neuropeptides.

Molecular size distribution of somatostatin-like immunoreactivity in the cerebroventricular fluid of neurosurgical patients.

The molecular size distribution of somatostatin-like immunoreactivity (SLI) in the cerebroventricular fluid of patients with Parkinson's disease, dystonic syndromes, multiple sclerosis, basal and midline tumors, epilepsy and pain syndromes was investigated by separation with a Sephadex G-50f column and subsequent radioimmunoassay of the eluate. Marked heterogeneity of SLI was observed in most of the pools investigated. The most conspicuous feature of the elution profiles was the preponderance of the peak coeluting with synthetic somatostatin-14, whereas the peaks comigrating with synthetic somatostatin-28 and attributable to precursor-like SLI represented only minor or trace amounts of total immunoreactivity. These findings are consistent with the greater biological activity of somatostatin-14 in the human central nervous system, whereas somatostatin-28 appears to represent the more active form in the pituitary and in the intestinal mucosa. Solely in the case of brain tumor patients, some differences could be seen, resulting in an approximately equal distribution of somatostatin-14 and somatostatin-28 in two pools of ventricular fluid and by the detection of a degradation product of somatostatin-14 in another one. These observations could be explained by a lowered barrier function as a consequence of increased intracranial pressure in case of brain tumors, which is well in accordance with a markedly elevated total protein content being a sign of a lowered barrier function.

Molecular size distribution of somatostatin-like immunoreactivity in the cerebrospinal fluid of patients with degenerative brain disease.

The molecular size distribution of somatostatin-like immunoreactivity (SLI) in the cerebrospinal fluid (CSF) of patients with brain disease was investigated by separation with a Sephadex G-25 superfine column and subsequent radioimmunoassay of the eluate. Marked heterogeneity of SLI in the CSF of control subjects as well as in demented patients, was observed. Controls and schizophrenics exhibited an SLI distribution pattern consisting mainly of two pronounced peaks: the first eluting with the void volume of the column; the second being compatible with a peptide of N-terminally extended somatostatin-14. SLI from the CSF of patients with senile dementia of the Alzheimer type (SDAT), multi-infarct dementia (MID) and normal pressure hydrocephalus (NPH) showed the same two peaks found in controls and schizophrenics; and in addition, a third peak co-eluting with somatostatin-14. However, this peak was more pronounced in patients with SDAT and MID than in patients with NPH. Re-chromatography of G-25 sf void volume immunoreactivity afforded two fractions of an apparent molecular weight of about 10,000 daltons and 15,500 daltons, respectively.

Permeability of epidural somatostatin and morphine into the intrathecal space of dogs.

In an in vivo saline perfusate of the intrathecal space of 6 dogs, the concentration of somatostatin was determined by radioimmunoassay before and over 2 h after epidural administration of 3 mg somatostatin. The total recovered amount of somatostatin was negligible, about 0.02%. However, within 50 min after the bolus epidural injection of somatostatin, the concentration per ml perfusate increased from 0.1 +/- 0.02 ng/ml to 138 +/- 102 ng/ml (P less than 0.001) and declined to 4 +/- 1.7 ng/ml after 120 min. This increase of the somatostatin concentration by 3 orders of magnitude might explain why epidurally administered somatostatin is effective in treatment of acute and chronic pain. In a control investigation with epidural morphine in another 6 dogs to prove the feasibility of the method, the total recovered amount of morphine in the intrathecal perfusate over 2 h was about 12%.

Cerebrospinal fluid immunoreactive substance P and somatostatin in neurological patients with peripheral and spinal cord disease.

We have measured substance P-like (SPLI) and somatostatin-like (SLI) immunoreactivities in cerebrospinal fluid of 49 patients with peripheral (polyneuropathy, lumboischialgia) and spinal cord disease and in 16 control patients. The patient groups showed significantly higher CSF SPLI levels than controls while the mean SLI levels were unchanged. Fractionated sampling of CSF (total volume 30 ml) in 20 patients with various neurological diseases showed no significant differences between early and late fractions for SLI. In contrast, lumbar-cisternal concentration gradients were negative for SPLI, total protein and IgG, and positive for the dopamine metabolite homovanillic acid and the serotonin metabolite 5-hydroxyindolacetic acid. This suggests that SPLI may be released into the lumbar CSF from lower levels of the neuraxis, presumably the spinal cord and spinal ganglia, whereas SLI stems from diffuse CSF secretion without spinal preponderance.

Ventricular somatostatin-like immunoreactivity in patients with basal ganglia disease.

The concentrations of somatostatin-like immunoreactivity (SLI) in lateral ventricular fluid of patients with extrapyramidal motor disease were determined by specific radio-immunoassay. Mean SLI levels were significantly lower in patients with Parkinson's disease (mean +/- SEM); 42.9 +/- 2.9 fmol/ml) and in patients with dystonic syndromes (39.4 +/- 3.2) than in patients with benign essential tremor (65.3 +/- 9.7). The lowest levels were found in patients with athetosis (34.7 +/- 5.4). In parkinsonian patients somatostatin levels correlated with the degree of akinesia, rigidity and autonomic disturbances.

Somatostatin-like immunoreactivity and substance-P-like immunoreactivity in the CSF of patients with senile dementia of Alzheimer type, multi-infarct syndrome and communicating hydrocephalus.

The concentrations of somatostatin-like immunoreactivity (SLI) and substance-P-like immunoreactivity (SPLI) in lumbar spinal fluid of patients with senile dementia of the Alzheimer type (SDAT), multi-infarct syndrome, communicating hydrocephalus and control patients were determined by specific radio-immunoassay. Mean SLI and SPLI levels were significantly lower in an aged control patient group (mean age 83.5 +/- 5.6 years) than in an adult control patient group (mean age 30.8 +/- 10 years). In the latter group SPLI levels correlated negatively with age. Mean SLI levels decreased with deterioration in SDAT patients by up to 33% in late dementia. SPLI correlated with SLI in SDAT patients but was decreased significantly only in late dementia patients. Moderate and insignificant decreases of SLI were observed in patients with multi-infarct syndrome or communicating hydrocephalus. Analysis of SLI by gel-permeation chromatography revealed molecular heterogeneity of SLI. At least four peaks of SLI were eluted, two of which had apparent molecular weights of about 10,000 and 15,500, possibly representing somatostatin precursors. The ratio of SRIF to SLI of higher molecular weight was increased in patients with dementia compared to control patients.

Separation of polyesters by gradient reversed-phase high-performance liquid chromatography on a 1.5 microm non-porous column.

Efficient separation of polyesters composed of a large number of individual oligomers was achieved on a 1.5 microm "non-porous" octadecylsilyl (ODS) silica support by gradient high-performance reversed-phase liquid chromatography (gRP-HPLC) with a mobile phase of acetonitrile, aqueous trifluoroacetic acid (0.2%) and tetrahydrofuran at ambient temperature and signal monitoring by UV absorption at 280 nm. Substantial signal splitting of oligomers in the low molecular weight (Mr) region is indicative that separation not only occurs with respect to molecular weight distribution (MWD) but also to chemical composition distribution (CCD) and functionality type distribution (FTD). Although separation according to CCD and FTD decreases with increasing number of oligomers, co-elution of species with identical number of repeat units but differing in either structure of repeat units or end-groups can be assumed from the relatively broad signals succeeding the aforementioned peaks showing at least partial resolution. Despite the observation that high Mr oligomers elute as sharp signals, the preceding observations suggest that each of these peaks presumably composes of more than one individual component. The polyester oligomers are eluted in the range of increasing Mr and therefore, either separation according to MWD or CCD/FTD was at least achieved for the low Mr sample constituents. Some principal mechanistic aspects of separation are discussed and adsorption seems to play the dominant role. The detection limit, defined as that sample amount yielding an unequivocal recognition on the base of its characteristic chromatographic fingerprint pattern was about 5,000 ppm for the pair Alftalat 3258 - Alftalat 3352 and 10,000 ppm for the pair Crylcoat 430 - Crylcoat 801.

Analysis of sulfonated compounds by ion-exchange high-performance liquid chromatography-mass spectrometry.

Ion-exchange high-performance liquid chromatography (HPIEC)-mass spectrometry (MS) was used for the analysis of different sulfonated compounds. HPIEC was performed on an aminopropyl column applying a gradient with increasing concentration of a buffer consisting of ammonium acetate-acetic acid and acetonitrile as the organic modifier. HPIEC is well suited to highly efficient separation of sulfonated compounds and furthermore, due to the volatility of ammonium acetate, the method is also appropriate for LC-MS coupling by the means of either atmospheric pressure chemical ionization or electrospray ionization. The applicability range of HPIEC-MS is demonstrated on the basis of a complex mixture of model substances consisting of sulfonated aromatics and textile dyes largely differing from each other in their structural properties.

High-performance liquid chromatography - atmospheric pressure negative chemical ionisation - mass spectrometry of 2,4-dinitrophenyl derivatives of amines.

High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) is used to detect 2,4-dinitrophenyl (DNP) derivatives of amines by means of negative chemical ionisation at atmospheric pressure. The high sensitivity and good comparability of UV- and MS-detection of DNP-derivatives of amines is shown by several examples. The high selectivity of the derivatisation and the detection method (UV and MS) is used for the analysis of unknown amines in aqueous phases after hydrolytic degradation of polyamide-amine- or polyamine-epoxide-adducts as well as for the characterisation of commercial products.

Somatostatin-like immunoreactivity in cerebroventricular fluid of patients with basal midline tumours.

The concentration of somatostatin-like immunoreactivity (SLI) was determined by specific radioimmunoassay in the cerebroventricular fluid of patients with tumours of the basal midline and compared to findings in patients with multiple sclerosis. Mean SLI levels of the two groups were not significantly different. In patients with basal tumours (astrocytoma, craniopharyngioma, etc.) SLI levels varied widely and tended to increase with increased intracranial pressure. Lateral ventricular SLI levels decreased in patients with blockade of foramen Monro and in patients with communicating hydrocephalus or post-radiation encephalopathy. There was no apparent correlation with dysfunction of the hypothalamohypophyseal axis.

Effects of haloperidol on somatostatin-like immunoreactivity in the CSF of Schizophrenic patients.

The levels of somatostatin-like immunoreactivity (SLI) were determined in the cerebrospinal fluid (CSF) of 14 schizophrenic patients before and after 3 weeks on haloperidol treatment. Baseline levels of SLI correlated negatively with psychopathological items on the Brief Psychiatric Rating Scale related to psychotic productivity. SLI levels increased after haloperidol treatment, but this increase did not correlate with psychopathological improvement. A difference in the ratio of larger and smaller molecular forms of the peptide was found before and after treatment. The drug-free samples showed a preponderance of the larger molecular forms, resulting in a ratio of 4:1, whereas the haloperidol-treated samples showed an equal distribution of both species.

Sample preparation, high-performance liquid chromatographic separation and determination of substance P-related peptides.

This review deals with the determination of low levels of substance P and peptide fragments derived from the undecapeptide, i.e. covers the whole amount of so-called substance P-like immunoreactivity (SPLI) in biological samples. First an overview of the most currently used sample pretreatment procedures is given, followed by a description of the most effective high-performance liquid chromatographic (HPLC) separation methods. Special attention is paid to the choice of the appropriate column and the possible pitfalls encountered in separation of fmol amounts of peptide material. Subsequently the most important techniques of detection are discussed. This section primarily focuses on the coupling of HPLC with radioimmunoassay (RIA), which is indispensable for detection of components in the fmol range at present. Finally, some aspects of preparation and chromatographic separation of radiolabelled antigens for use in RIA are discussed.

Labeling of insulin with non-radioactive 127I and application to incorporation of radioactive 125I for use in receptor-binding experiments by high-performance liquid chromatography.

Conditions for the labeling of insulin with radioactive iodine isotopes were investigated by means of incorporation of non-radioactive 127I into the peptide. Either the chloramine-T (CT) or lactoperoxidase-hydrogen peroxide (LPO) technique was applied and reversed-phase high-performance liquid chromatography (RP-HPLC) was used for analysis of the reaction products. The LPO method provided the 127I-labeled peptide within 15-30 min, whereas the CT alternative yielded the labeled substrate even within 15 s. However, the latter reaction can only be controlled in a reproducible manner with difficulty and undesired side-reactions became increasingly prominent when the reaction time of 15 s was exceeded for only a few seconds. In another experiment, the LPO technique was applied for radiolabeling insulin with 125I. The product was first purified by size-exclusion chromatography (SEC) and then subjected to RP-HPLC. SEC yielded two peaks. The smaller one, which eluted at a slightly higher Kd value (accounting for about 14% of total radioactivity) predominantly consisted of material eluting at the column's void volume under the conditions of RP-HPLC, whereas the main SEC fraction (accounting for about 86% of total radioactivity) yielded a single peak, as shown by HPLC. The radioactive material attributable to the main SEC fraction revealed the expected receptor-binding properties, as evidenced by displacement experiments with non-radioactive insulin, as well as the action of tetradecanoyl phorbol acetate on the binding characteristics and thus indicating formation of a labeled hormone retaining biological activity.

Marked improvement of a substance P radioimmunoassay by reduction of 125I-labelled [Tyr8]-substance P prepared by the chloramine-T method with mercaptoethanol and subsequent purification by reversed-phase liquid chromatography.

[Tyr8]-substance P was radiolabelled with 125I by the application of the chloramine-T method. Due to the high oxidative potential of the 125I-chloramine-T system the purified reaction product was converted into a derivative, which presumably had been oxidized to the corresponding sulphoxide at Met11. This conversion was shown by reversed-phase high-performance liquid chromatography after consecutive reduction-oxidation experiments of the freshly prepared radiopeptide. The oxidized derivative exhibited only negligible binding to substance P receptors isolated from rat brain homogenates. However, in contrast, it showed marked cross-reaction to the antibody raised in rabbits against synthetic SP(1-11). The variance in the quantification of identical samples was marked, and the measurement of concentrations in the lower pg/ml range was not sensitive enough to determine levels of substance P-like immunoreactivity in human cerebrospinal fluid. Assay sensitivity could be substantially improved and variance significantly decreased by the use of a radiopeptide, which had been labelled by the chloramine-T method and which had been subsequently reduced with mercaptoethanol and purified by liquid chromatography.

Preparation and chromatographic purification of 125I-[Tyr0]-somatostatin-14 for the use in radioimmunoassay and receptor-binding experiments.

[Tyr0]-somatostatin-14 (SST-14) was converted to the corresponding radiolabeled 125I-[Tyr0]-SST-14 derivative by use of the chloramine-T technique. After solid-phase extraction (SPE) of the crude radiopeptide with microfine silica gel and desorption with acetone-water-acetic acid 39:40:1, the label was subjected to size exclusion chromatography (SEC) on Sephadex G-25 fine. Fractions attributable to the target compound were collected and investigated with respect to their specific as well as non-specific binding to a specific anti-somatostatin antibody. All fractions exhibiting an optimum ratio of specific versus non-specific binding were pooled and lyophilized for their further use in both radioimmunoassay (RIA) measurements of somatostatin like immunoreactivity (SLI) and receptor-binding experiments. A specific activity of approx. 1.6 x 10(6) Ci/M was calculated for the radiolabel prepared in this manner. Approximately 85-90% of radioactivity attributable to labeled [Tyr0]-SST species was incorporated into the desired mono-iodinated component. When 125I-[Tyr0]-SST-14 was purified by reversed-phase high performance liquid chromatography (RP-HPLC) using isocratic elution with 0.1 M triethylammonium formate (TEAF) buffer of pH 2.2 in 22% acetonitrile after prior SPE, specific binding decreases to about 80% compared with the value obtained for the radiopeptide subjected to SEC. Nevertheless, RP-HPLC proves as an efficient tool for rapid purity control of 125I-[Tyr0]-somatostatin-14 samples at different storage time intervals.

Application of [(125)I]-[Tyr8]-substance P prepared by the chloramine-T method to receptor-binding experiments after subsequent reduction with mercaptoethanol and purification by reversed-phase liquid chromatography.

Radiolabeling of [Tyr8]-substance P ([Tyr8]-SP) with the (125)I-isotope was performed by use of the chloramine-T technique. The primary formed radiolabeled product, having been quantitatively converted to the corresponding sulfoxide yielding [(125)I]-[Tyr8]-(Met11-->O)-SP completely lacked any binding to proteins rich in SP receptor populations. However, after reductive treatment with mercaptoethanol for about 2 h, a complete reconstitution of the Met11 thioether structure was observed. The reduced peptide, consisting of [(125)I]-[Tyr8]-(Met11)-SP was separated from its by-products by reversed-phase high-performance liquid chromatography on octadecylsilyl silica gel with 100 mM triethyl ammonium formate buffer containing 22% acetonitrile (pH 2.2). The labeled SP derivative prepared by this two-step synthesis was obtained in 73% overall yield related to the [Tyr8]-SP starting material and exhibited a specific activity of 1.9-10(6) Ci/M. In contrast to [(125)I]-[Tyr8]-->(Met11-->O)-SP, satisfactory receptor-binding was now observed with the [(125)I]-->[Tyr8]-(Met11)-SP derivative.

Recovery of substance P and related C-terminal fragments on solid-phase extraction cartridges for subsequent high-performance liquid chromatographic separation and radioimmunoassay.

The recoveries of substance P (SP) and five related peptides were evaluated on different types of solid-phase extraction sorbent. Best results were obtained by use of a C18 silica gel cartridge. Marked differences of extraction yields occurred for the different peptide fragments and, in general, recovery increased with increasing hydrophobicity of the peptide when reversed-phase materials like C18 and C8 cartridges were used. This observation is indicative of a sorption-desorption mechanism by prevailing solvophobic interactions. A similar trend was found when phenylpropyl silica gel (CPhenyl), generally known as a reversed-phase adsorbent of lower hydrophobicity, was used. It was concluded that a substantial participation of analyte-matrix pi-pi interactions has to be taken into account when extraction yields are compared with corresponding values obtained by use of a C8 cartridge. With CN silica gel cartridges, marked differences in extraction yields were obtained by use of acetonitrile or methanol as the organic modifier. As an attempt to explain this observation, conformational effects were assumed for the sorption-desorption behaviour of the peptides on the polar matrix.

Determination of the retinoid (E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2- phenylethenyl)naphthalene and its phenolic metabolite in human plasma by reversed-phase high-performance liquid chromatography.

A rapid, sensitive and specific high-performance liquid chromatographic assay was developed for the determination in plasma of (E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2- phenylethenyl)naphthalene (1) and its phenolic metabolite (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- methylethenyl]-phenol (2). An extraction procedure using protein precipitation and liquid-liquid extraction was combined with a simple column-switching technique. To minimize sample consumption, the assay was adapted to a sample volume of 200 microliters, which was sufficient for more than 90% of all determinations. The quantification limit was 100 ng/ml for 1 and 2, whereas the detection limits were 20 and 30 ng/ml, respectively. The recoveries for 1 and 2 were 91 and 94%, respectively, using ultraviolet detection at 280 nm. The assay was used to quantify both compounds in human plasma samples.

Long-term recovery of superficial and deep glomeruli after acute renal failure evoked by warm ischemia.

A study was made on the number of glomeruli in the outer cortex, midcortex, and inner cortex 4 weeks after 45 min of warm ischemia, using alcian blue to stain functioning glomeruli. During this time the stainable glomeruli in the kidney as a whole had decreased in number from 32,000 to 16,800. The superficial glomeruli were reduced by only about 30%, whereas in the juxtamedullary zones the number of functioning nephrons were reduced by 80%. This shows that the juxtamedullary nephrons are the most susceptible to degeneration in the recovery phase of acute renal failure caused by ischemia. The decrease in the number of glomeruli was accompanied by a reduction in total glomerular filtration rate to about the same extent. In contrast, the kidney weight was reduced by only about 20%, suggesting compensatory hypertrophy of the remaining tubules. Fluid reabsorption, urine osmolality, and potassium-secreting ability also remained decreased. The contralateral kidney responded with a compensatory hypertrophy and an increase in glomerular filtration rate, whereas the number of glomeruli in the cortex as a whole and in the individual cortical zones in this kidney remained the same as in control kidneys. It is suggested that the trapping of red cells in the juxtamedullary circulation seen soon after restoration of circulation to kidneys subjected to warm ischemia will also cause a further degeneration of the juxtamedullary nephrons.