Transcobalamin II in human seminal plasma.

Study of cobalamin-binding proteins revealed seminal plasma to be the most concentrated site of transcobalamin II in man. The next richest normal fluid, blood, has approximately one-tenth its concentration. Normal seminal unsaturated cobalamin-binding capacity averaged 15,030 +/- 7,290 pg/ml, of which 11,550 +/- 6,660 pg/ml was transcobalamin II. Transcobalamin II levels were markedly diminished in subjects lacking seminal vesicles (1520-1660 pg/ml), but not after vasectomy. This suggests that seminal vesicles are the chief source of this protein in semen. R binder concentration was increased in postvasectomy subjects (9,970 +/- 4,900 pg/ml vs. 2,980 +/- 1,370 pg/ml in normals) and varied in other patients. The endogenous cobalamin content of semen was only 88-699 pg/ml, and was carried largely by R binder rather than by transcobalamin II. The function of the unusually large seminal transcobalamin II pool in reproduction is unknown, but seems unlikely to be related solely to cobalamin transport needs, at least within the male reproductive tract itself.     

The metabolism in man of intravenously injected iodine-labelled trascobalamin I not saturated with cobalamin.

Transcobalamin I not saturated with cobalamin was purified. After iodine-labelling, a fraction of approximately 0.6 retained its cobalamin-binding capacity. Iodinated protein was used for turnover studies in three healthy subjects. The plasma curves obtained were identical to those of iodinated transcobalamin I saturated with cyanocobalamin. The cobalamin-binding capacity of the iodinated protein was determined on microcolumns containing insolubilized cobalamin. The binding capacity decreased rapidly, only 50% of the initial unsaturated cobalamin-binding capacity remaining after 0.2 to 0.3 days. It is concluded that the turnover of transcobalamin I not saturated with cobalamin does not differ from the turnover of transcobalamin I saturated with cyanocobalamin, and that transcobalamin I saturated with cobalamin in vivo is metabolized at the same rate as is transcobalamin I saturated with cyanocobalamin in vitro.     

Lecithin:Cholesterol Acyl Transfer in Plasma of Normal Persons in Relation to Lipid and Lipoprotein Concentration

Gimsing, P. &; Nexö, E. The Metabolism in Man of Intravenously Injected Iodine-Labelled Transcobalamin I Not Saturated with Cobalamin. Scand. J. clin. Lab. Invest. 36, 669–672, 1976.

Transcobalamin I not saturated with cobalamin was purified. After iodine-labelling, a fraction of approximately 0.6 retained its cobalamin-binding capacity. Iodinated protein was used for turnover studies in three healthy subjects. The plasma curves obtained were identical to those of iodinated transcobalamin I saturated with cyanocobalamin. The cobalamin-binding capacity of the iodinated protein was determined on microcolumns containing insolubilized cobalamin. The binding capacity decreased rapidly, only 50% of the initial unsaturated cobalamin-binding capacity remaining after 0.2 to 0.3 days. It is concluded that the turnover of transcobalamin I not saturated with cobalamin does not differ from the turnover of transcobalamin I saturated with cyanocobalamin, and that transcobalamin I saturated with cobalamin in vivo is metabolized at the same rate as is transcobalamin I saturated with cyanocobalamin in vitro.     

Unsaturated and cobalamin saturated transcobalamin I and II in normal human plasma

Insolubilized antibody against human transcobalamin I has been used as a specific and precise tool for the separation of transcobalamin I (and III) from transcobalamin II. Range and mean (in parentheses) for the unsaturated binding capacity for twenty samples are 40-190 (90) pmol/l and 220-1170 (560) pmol/l for transcobalamin I (and III) and transcobalamin II, respectively. The similar figures for the cobalamin saturated transcobalamins are 200-549 (320) pmol/l and 75-475 (160 pmol/l. On analyses of the cobalamins attached to each of the transcobalamins, it is shown that methylcobalamin accounts for most of the cobalamins attached to transcobalamin I whereas transcobalamin II carries most of the 5'-deoxyadenosylcobalamin.     

Clinical relevance of the quantification of apolipoprotein E in cerebrospinal fluid

Apolipoprotein E was measured in paired sera and cerebrospinal fluid samples from 483 neurological patients. The average apolipoprotein E concentration was 7.5 (+/- 3.1) mg/l in cerebrospinal fluid and 93.5 (+/- 29.7) mg/l in serum. Mean apolipoprotein B concentrations in 88 patients were 0.77 +/- 3.4 mg/l in cerebrospinal fluid and 1.06 +/- 0.31 g/l in serum. The apolipoprotein E concentration in cerebrospinal fluid was much greater than expected for passive diffusion from serum. By contrast to apolipoprotein B, there was no correlation between the apolipoprotein E cerebrospinal fluid/serum concentration quotient and the albumin concentration quotient. This suggests that apolipoprotein E in cerebrospinal fluid, unlike apolipoprotein B, is locally synthesized and that the use of a cerebrospinal fluid/serum concentration quotient is unnecessary. In a control group (n = 64) the mean cerebrospinal fluid apolipoprotein E value (+/- SD) was 5.9 (1.6) mg/l. Elevated cerebrospinal fluid apolipoprotein E concentrations were observed in acute (n = 22, 12.5 +/- 6.2 mg/l) and chronic inflammatory central nervous system diseases (n = 15, 10.4 +/- 2.4 mg/l).     

Characterization of a Cobalamin-binding Plasma Protein from a Patient with Hepatoma

A cobalamin-binding protein has been purified by affinity chromatography from plasma of a patient with hepatoma and a 10,000-fold increase in the concentration of the plasma cobalamin-binding capacity. The protein behaved as normal transcobalamin I in gel filtration, agar gel electrophoresis, im-munoelectrophoresis, precipitation by ammonium sulphate, and cobalamin-binding studies. The protein contained 38 per cent carbohydrate, and the relative molecular mass based on amino acid and carbohydrate analyses was 69,000. The molar absorption coefficient of cyanocobalamin bound to the protein was determined to be 36,000 at 362 nm. On amino acid sequencing, one amino terminal was found, and the first 13 residues were determined as Glu-Ile-Ser/Cys-Glu-Val-Ser/Cys-Glu-Glu-Asx-Tyr-Ile-Arg-Leu/Ile.     

Cerebrospinal fluid levels of catechols in patients with neurogenic orthostatic hypotension

In multiple system atrophy (MSA) and pure autonomic failure (PAF), orthostatic hypotension (OH) results from deficient noradrenaline release from sympathetic nerves during standing. Post-mortem findings have indicated loss of central noradrenergic cells in both diseases. The present study sought in vivo neurochemical evidence for central noradrenergic deficiency in patients with OH due to MSA or PAF. A total of 28 patients with OH (18 with MSA; 10 with PAF) had cerebrospinal fluid and blood sampled for levels of noradrenaline and its neuronal metabolite dihydroxyphenylglycol. A control group of 44 subjects included 10 elderly normal volunteers, 10 patients with Alzheimer's disease, 18 patients with dysautonomia (postural tachycardia syndrome or neurocardiogenic syncope) and six patients with MSA in the absence of OH. Patients with OH had lower cerebrospinal fluid concentrations of noradrenaline (0.53+/-0.07 nmol/l) and dihydroxyphenylglycol (6.52+/-0.46 nmol/l) than did control subjects (0.90+/-0.09 and 9.64+/-0.46 nmol/l respectively; P =0.0001). The MSA+OH group had higher plasma levels of both catechols (noradrenaline, 1.31+/-0.16 nmol/l; dihydroxyphenylglycol, 5.08+/-0.43 nmol/l) than did the PAF group (noradrenaline, 0.38+/-0.08 nmol/l; dihydroxyphenylglycol, 2.53+/-0.30 nmol/l; P <0.001), despite similarly low cerebrospinal fluid levels. Among MSA patients, those with OH had lower cerebrospinal fluid levels of noradrenaline and dihydroxyphenylglycol than those without OH (noradrenaline, 1.71+/-0.64 nmol/l; dihydroxyphenylglycol, 10.41+/-1.77 nmol/l respectively; P =0.006). The findings are consistent with central noradrenergic deficiency in both MSA+OH and PAF. In MSA, central noradrenergic deficiency seems to relate specifically to OH.     

Concentration and physicochemical characterisation of unsaturated cobalamin binding proteins in amniotic fluid

Indirect evidence for the presence of intrinsic factor in amniotic fluid has been provided recently, using either a radioisotope binding assay or a radioimmunoassay. We have determined the unsaturated cobalamin binding capacity and the physicochemical properties of the 3 cobalamin binding proteins in 59 amniotic fluids using radioisotope binding assay, gel filtration and isoelectrofocussing. A good correlation with gestational age was found for the total unsaturated Cbl binding capacity (r = 0.735) and for the concentration of unsaturated haptocorrin (r = 0.746), but not for the concentration of unsaturated intrinsic factor (r = 0.003). When their binding capacities were expressed as a percentage of the total unsaturated Cbl binding capacity, the percentage of intrinsic factor, transcobalamin II and transcobalamin III (the less acidic fraction of haptocorrin) decreased and the percentage of haptocorrin increased in function of gestational age. The physicochemical properties of intrinsic factor in amniotic fluid were close to those in gastric juice: the molecular mass was estimated to 49,200 +/- 4,900 Da (n = 24) in Sephacryl S 300 gel filtration, the cobalamin-protein complex was resolved in 2-6 isoproteins isoelectric at a pH range of 4.6-5.8 and with a mean isoelectric point of 5.18 +/- 0.16 (n = 5) in isoelectrofocusing and it crossreacted with anti-intrinsic factor autoantibodies (from a Biermer anaemia serum). Amniotic fluid collected at 13 wk of gestational age was found to contain intrinsic factor and haptocorrin with less acidic isoproteins than those usually observed in gastric juice and serum. This could indicate that sialic acid is less involved in the composition of the carbohydrate core of cobalamin binding glycoproteins in this period of the gestational age than later on and that cobalamin binding proteins have mainly a foetal origin.     

Determination of gangliosides and sulfatide in human cerebrospinal fluid with a microimmunoaffinity technique

An immunostaining procedure has been developed for the assay of the gangliotetraose gangliosides and sulfatide in cerebrospinal fluid. Gangliosides of the gangliotetraose series were individually determined with cholera toxin B-subunit (CT-B) and an anti CT-B monoclonal antibody after chromatography and sialidase hydrolysis to GM1 on high performance thin-layer plates. Sulfatide was determined by thin-layer chromatography using an anti-sulfatide antibody. The method was applied to normal cerebrospinal fluid from 20 adults and 30 children. The concentration of the gangliotetraose series gangliosides in adults varied from 100-300 nmol/l with a mean value of 230 +/- 56 nmol/l. Corresponding values for sulfatide were 30-225 nmol/l and 140 +/- 46 nmol/l. The values for gangliosides and sulfatide in children increased during development. The major gangliosides in cerebrospinal fluid of adults were GT1b and GD1b and in children GD1a and GT1b.     

Application of a simple immunoadsorption assay for the measurement of saturated and unsaturated transcobalamin II and R-binders

In this paper an immunoadsorption method for the selective measurement of the concentrations of saturated and unsaturated transcobalamin II and R-binders in human plasma is presented. With this assay the concentrations of saturated transcobalamin II found in the plasma of 70 normal individuals ranged from 20-220 pmol per litre, with a mean of 70 pmol/l. The concentration of R-binders, carrying cobalamin, ranged from 87-491 pmol/l (mean 195 pmol/l). The 33-203 pmol/l (mean 111 pmol/l) of cobalamin analogues were found to be almost exclusively bound to the R-binder fraction. The level of saturated binding proteins is not, or only marginally, influenced by the absorption of ingested cobalamin, even with an oral dose of 15 micrograms. The concentration of transcobalamin II-bound cobalamin is apparently not determined by the availability of unsaturated binding protein. On the contrary, a positive correlation was found between the R-binder-cobalamin concentration and total R-binder level. These observations suggest that the concentration of transcobalamin II-bound cobalamin is primarily determined by the concentration of cobalamin in the tissues.     

Characterization of a cobalamin-binding plasma protein from a patient with hepatoma.

A cobalamin-binding protein has been purified by affinity chromatography from plasma of a patient with hepatoma and a 10,000-fold increase in the concentration of the plasma cobalamin-binding capacity. The protein behaved as normal transcobalamin I in gel filtration, agar gel electrophoresis, immunoelectrophoresis, precipitation by ammonium sulphate, and cobalamin-binding studies. The protein contained 38 per cent carbohydrate, and the relative molecular mass based on amino acid and carbohydrate analyses was 69,000. The molar absorption coefficient of cyanocobalamin bound to the protein was determined to be 36,000 at 362 nm. On amino acid sequencing, one amino terminal was found, and the first 13 residues were determined as Glu-Ile-Ser/Cys-Glu-Val-Ser/Cys-Glu-Glu-Asx-Tyr-Ile-Arg-Leu/Ile.     

Measurement of transcobalamin by ELISA

BACKGROUND: Transcobalamin is essential for the cellular internalization of cobalamin. Methods to quantify the unsaturated protein are available, but few attempts have been made to develop methods to quantify the sum of unsaturated and cobalamin saturated transcobalamin. METHODS: gamma-Globulins from two polyclonal rabbit antibodies against recombinant human transcobalamin were used as capture and detection antibodies, and recombinant human transcobalamin was used as calibrator in an ELISA design. RESULTS: The ELISA is specific for transcobalamin and has a detection limit of <1.6 pmol/L. The imprecision (CV) is 4-6% for mean concentrations of 13-70 pmol/L. The central 95% interval for serum from healthy blood donors (n = 77) was approximately 600-1500 pmol/L and showed limited variation with age and sex. No correlation was observed between the marker of acute phase reaction, C-reactive protein, and transcobalamin in plasma. CONCLUSIONS: The ELISA measures total transcobalamin in serum and thus can be used for measurement of transcobalamin in patients treated with cobalamin.     

Measurement of 5-aminolaevulinic acid by reversed phase HPLC and fluorescence detection

A new method for sensitive measurement of delta-aminolaevulinic acid (ALA) in biological material is described. ALA is derivatized with dansyl chloride, separated by HPLC and estimated using a fluorescence detector. The pretreatment of biological samples includes desamination of L-alpha-aminoacids with L-aminoacid-oxidase before dansylation. The sensitivity of the method is slightly below 1 pmol/injection for standards and the lower limit of quantification is 0.1 mumol/l for plasma and 10 nmol/l for cerebrospinal fluid. Reference values in plasma are 3.53 +/- 1.75 (SD) (n = 43) mumol/l and in packed erythrocytes they ranged from 6 to 26 mumol/l (mean: 14.0 +/- 5.5 mumol/l). In cerebrospinal fluid of non-porphyric individuals less than 2 nmol/l were recovered.     

Effect of meningitis and probenecid on the penetration of vancomycin into cerebrospinal fluid in rabbits.

This study examined the effects of experimental pneumococcal meningitis and probenecid administration on the penetration of parenterally administered vancomycin into cerebrospinal fluid in rabbits. Bacterial killing was also examined in infected animals. Meningitis was induced by intracisternal inoculation of Streptococcus pneumoniae. Vancomycin was administered in a loading dose followed by a continuous intravenous infusion for 6 h. Serum and cerebrospinal fluid samples were obtained at 0, 2, 4, and 6 h for antibiotic assays and quantitative cultures. Meningitis significantly enhanced the penetration of vancomycin into cerebrospinal fluid, but probenecid administration had no effect. In normal rabbits, at 6 h the mean percent penetration (cerebrospinal fluid concentration/serum concentration x 100%) +/- the standard deviation was 1.9 +/- 0.9% in the nonprobenecid group (n = 10) and 1.9 +/- 1.1% in the probenecid group (n = 9). In rabbits with experimental pneumococcal meningitis, the mean percent penetration at 6 h was 3.9 +/- 2.6% in the nonprobenecid group (n = 11) and 4.3 +/- 2.1% in the probenecid group (n = 9). Mean bacterial titers in the cerebrospinal fluid of infected animals decreased by more than 3.0 log 10 colony-forming units per ml in both the nonprobenecid and the probenecid groups.     

Pharmacokinetics of cefamandole and ampicillin in experimental meningitis.

The penetration of cefamandole and ampicillin into the cerebrospinal fluid of rabbits with and without pneumococcal meningitis was evaluated. In normal animals, a mean maximum concentration of 0.22 +/- 0.13 microgram of cefamandole per ml was measured in the spinal fluid after a dose of 150 mg/kg given intramuscularly; with 25 and 50 mg/kg doses, no antibiotic was detected in the cerebrospinal fluid. With ampicillin, in intramuscular doses of 200 and 300 mg/kg, the mean maximum concentrations encountered in the cerebrospinal fluid were 1.59 +/- 0.4 and 1.47 +/- 0.44 microgram/ml, respectively. Penetration of cefamandole, and to a lesser extent ampicillin, was increased after 24 h of experimental meningitis. With cefamandole, the concentration of drug in the cerebrospinal fluid exceeded the usual inhibitory concentration for Haemophilus influenzae only with the 150 mg/kg dose. After 48 h of meningitis, there was a trend toward higher levels of antibiotic in the cerebrospinal fluid, but the difference between animals infected 24 versus 48 h was not statistically significant. In animals with meningitis, serum concentrations after 150 mg of cefamandole per kg and both ampicillin doses studied were 32 to 38% lower than the serum levels achieved in normal rabbits after identical doses of antibiotic.     

An improved specific and sensitive radioenzymatic method for determination of serotonin concentrations in biological fluids (radioenzymatic serotonin method)

Existing radioenzymatic assays for determining the concentration of serotonin in biological fluids have been further modified resulting in increased sensitivity and a lowered detection limit (0.017 pmol). The assay has been used for determinations in platelet-poor plasma (3.3-20.5 nmol/l) and cerebrospinal fluid (0.15-20 nmol/l) from man.     

delta-Aminolaevulinic acid in plasma, cerebrospinal fluid, saliva and erythrocytes: studies in normal, uraemic and porphyric subjects

delta-Aminolaevulinic acid (ALA) was determined by g.l.c. with electron-capture detection. Normal plasma level was 92 nmol/l (SD = 39, n = 89, range 24-270 nmol/l). ALA was undetectable in 35 of 53 samples of normal cerebrospinal fluid (limit of assay 2 nmol/l). The mean of the other 18 samples was 19 nmol/l (SD = 10, range 6-36 nmol/l). Salivary ALA was generally only 10-30% of the plasma level in normal and porphyric subjects. Erythrocytes of normal and porphyric subjects contained no detectable ALA and were impermeable to its entry. ALA clearance correlated closely with that of creatinine, consistent with it being excreted by glomerular filtration with limited tubular reabsorption. In chronic renal failure, serum ALA was elevated to a maximum of three to four times the normal, but its urinary excretion was reduced, in keeping with lessened production. In two cases of acute intermittent porphyria with overwhelming neuropathy the maximum plasma levels of ALA were 9 and 12 mumol/l. Haematin infusion decreased the ALA levels but without obvious clinical benefit. Limited neurological recovery occurred without major reduction in plasma levels of ALA. One subject's attack was precipitated by pregnancy. The neonate was apparently normal, despite high levels of ALA in maternal plasma throughout gestation and a high level of ALA in the cord blood. The observations described here do not support the view that ALA may be directly neurotoxic.     

Automated assay of gamma-aminobutyric acid in human cerebrospinal fluid

We describe an automated amino acid analyzer with fluorescence detection (o-phthalaldehyde) which permits sensitive and rapid determinations of gamma aminobutyric acid in human cerebrospinal fluid. Concentrations as low as 50 nmol/liter can be accurately determined in 100 mul samples at the rate of one sample per hour. Concentrations in untreated cerebrospinal fluid increase rapidly after sampling by lumbar puncture. The concentration in immediately deproteinized samples from 38 patients with intervertebral disc disorders was 220 +/- 81 nmol/liter (mean +/- SD).     

Simultaneous determination of tryptophan and its 5-hydroxy metabolites in human cerebrospinal fluid by reversed phase liquid chromatography with electrochemical detection

A sensitive ion-pair reversed phase liquid chromatographic method for determining tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine (serotonin) and 5-hydroxyindole acetic acid has been developed. To separate these indoles in cerebrospinal fluid both octadecyl- and phenyl-bonded columns were tested. The effect of an ion-pair reagent (n-heptyl sulphonate) and pH of the mobile phase on the separation was examined. With the method described the concentrations of the four indoles can be determined within 45 min without sample pretreatment. The sensitivity obtained with electrochemical detection is 0.45 mumol/l for tryptophan and 2-4 nmol/l for 5-hydroxyindoles in human cerebrospinal fluid samples.