Ole Svensmark

Aspects of the Pharmacology of Phenytoin (Dilantin®) and Phenobarbital Relevant to their Dosage in the Treatment of Epilepsy

Findings are reviewed concerning the distribution and fate of phenytoin and pheno‐barbital in animals and in man. The serum concentration of these drugs can be considered an adequate expression of their concentration in the brain and hence of their anticonvulsant effect.

Accumulation of Phenobarbital in Man

Equations relating serum concentrations of phenobarbital, dose/kg body weight, and rate of elimination were derived. Agreement between calculated and observed concentration‐time curves was obtained in 5 of 6 subjects, and between calculated and observed ratios of level to dose in 18 patients. The average ratio of serum levels to dose/kg body weight was 10.0 μg/ml per mg/kg. The average rate of elimination was 13.3 %/24 h, in agreement with previous findings. From the rates of elimination the time to 95% level was calculated to be 2–4 weeks with an average of 3 weeks. It is suggested that the dose of phenobarbital be doubled during the first 4 days of medication, thereby reducing the time to level to about 3 days.

Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography.

1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.

Isoelectric point of native and sialidase-treated human-serum cholinesterase

1. 1. The electrophoretic mobility of native and sialidase-treated human-serum cholinesterase was determined by paper electrophoresis over the pH range 2.8–9.6; the determination included corrections for electro-osmosis, evaporation, apper-structure and adsorption of protein to the paper. The performance of the method was controlled with proteins of known mobility, orosomucoid and coeruloplasmin. 2. 2. The isoelectric point of native cholinesterase was found to be 2.9–3.0 and that of sialidase-treated cholinesterase 6.7–7.0. This is an agreement with previous findings that human-serum cholinesterase contains a large number of sialic acid residues in a terminal position. 3. 3. At pH 8.6 the electrophoretic mobility of native cholinesterase was −3.1·10−5 cm2/V/sec and that of sialidase-treated cholinesterase −0.2·10−5 cm 2/V/sec. 4. 4. Between pH 4 and 9.6 the mobility of sialidase-treated cholinesterase was 2.9·10−5 units above that of native cholinesterase. 5. 5. At pH 2.8 this difference decreased to about 2·10−5 units. 6. 6. It is suggested that the mobility-pH curve might be of value in the differentiation between closely related types of cholinesterases.

CARBOXYLESTERASES WITH DIFFERENT SUBSTRATE SPECIFICITY IN HUMAN BRAIN EXTRACTS

Abstract— Methods for the determination of carboxylesterase activity in soluble as well as in particulate samples with p‐mtrophenylacetate and ‐butyrate and α‐naphthylacetate and ‐butyrate as substrates are described. Of the carboxylesterase activity of human brain, 8‐20% was present in aqueous extracts. Particle‐bound carboxylesterases could not be solubilized. By DEAE‐cellulose chromatography the carboxylesterases were separated into 6 more or less inhomogeneous fractions. One of these was further resolved into 2 fractions by chromatography on CM‐cellulose. Fractions obtained by ion exchange chromatography were resolved into several fractions by isoelectric focusing. Gel chromatography on Sephadex G‐200 resolved the carboxylesterases of brain extract into two fractions (molecular weights about 60.000 and 300,000). At least 4 different types of carboxylesterases could be distinguished on the basis of different substrate specificity.

Human red cell acid phosphatase: purification and properties of the A, B and C isozymes

Human red cell acid phosphatase isozymes encoded by three alleles (ACP1*A, ACPI*B and ACP1*C), each of which generates two isozymes, (f) and (s), were purified to homogeneity. The molecular mass of the six isozymes (Af, As, Bf, Bs, Cf and Cs) was estimated to be 17-18 kDa, the mass of the f isozymes probably being slightly higher than that of the s isozymes. It was indicated that the isozymes react with p-nitrophenyl phosphate in the mono anionic state, and that a group with a pKa value of about 6, which may be histidine, is of importance for the catalytic function of the s isozymes. Significant differences between the f and s isozymes were observed with respect to specific activity. Km (p-nitrophenyl phosphate), Ki (p-aminobenzylphosphonic acid), amino acid composition, stability in the presence of urea, thermal stability, retention time in size-exclusion chromatography of the native isozymes and migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis, In contrast, identical or similar properties were observed for the three genetically different f isozymes, and the same was the case for the three s isozymes. It is suggested that the f and s isozymes serve different functions in the cell.

Multiple Forms Of Soluble Butyrylcholinesterase In Human Brain

Summary 1. 30-55% of the butyrylcholinesterase (EC 3.1.1.8) activity of human brain was present in an aqueous extract. Particle-bound butyrylcholinesterase could not be made soluble. 2. In DEAE-cellulose chromatography soluble butyrylcholinesterase was separated in one inhomogeneous main fraction and several minor fractions. Gel chromatography on Sephadex G-200 resolved the main fraction into two inhomogeneous fractions, one of higher molecular weight (300 000-400 000), the other of lower molecular weight (50 000-150 000). 3. Electrofocusing of the butyrylcholinesterase fraction of higher molecular weight revealed two inhomogeneous fractions in the pH ranges 3.8-4.7 and 5.6-8.2. By treatment with neuraminidase (EC 3.2.1.18) the most acid subfractions disappeared and alkaline subfractions appeared. The acid and the most alkaline subfractions may originate from plasma trapped in the tissue. Electrofocusing of the fraction of lower molecular weight showed at least 6 subfractions in the pH range 5.6-8.0. Treatment with neuraminidase had no effect on their isoelectric points. 4. Brain tissue thus contains at least 4-5 soluble butyrylcholinesterase fractions of high molecular weight with isoelectric points ranging from 5.6 to 7.0 and at least 6 low-molecular-weight fractions with isoelectric points from 5.6 to 8.0.

Statistical analysis of the measurement errors in the determination of fragment length in DNA-RFLP analysis.

DNA from human whole blood samples was digested with the restriction enzyme HinfI and RFLP analysis performed using the single locus probes MS1, MS31, MS43a and YNH24. The intergel variation of 3291 duplicate measurements of fragment lengths in terms of basepairs was investigated. The difference between two measurements of the same fragment on different gels increased approximately exponentially with increasing fragment length. After transformation of the fragment length into a normalized migration distance it was found that the difference between two transformed measurements was normally distributed with a S.D. (0.70 mm) which was independent of the fragment length. The errors of band 1 and band 2 on the same lane were correlated (r2 = 0.8). It is useful in the calculation of frequencies and in retrieval procedures and also in the calculation of likelihood ratios to be able to use a S.D. which is independent of the fragment length.

Carboxylesterases of human brain extract. Purification and properties of a butyrylesterase.

1. A carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) from human brain extract was prepared to purity using DEAE-cellulose, Sephadex G-200, and fractionation with (NH4)2SO4. The yield was about 20%. 2. Esters of butyric acid were split faster than esters of acetic, propionic and valeric acid, and the enzyme is tentatively designated as a butyrylesterase. Thiocholine esters were split at low rates. 3. The molecular weight was estimated as 340 000 using gel chromatography on Sephadex G-200. In isoelectric focussing the enzyme was resolved into several peaks (pI 4.0--4.7). The low isoelectric point does not seem to be due to terminal sialic acid residues. 4. The enzyme was irreversibly inhibited by diethyl-p-nitrophenyl phosphate (ki = 206 mol-1 - 1 - s-1) and by diisopropylfluorophosphate. The carboxylesterase inhibitor bis-p-nitrophenyl phosphate and eserine did not inhibit the enzyme. 5. The enzyme was progressively inhibited by p-hydroxy-mercuribenzoate, and reactivated by dithiothreitol and 2-mercaptoethanol. N-Ethylmaleimide inactivated the enzyme very slowly, whereas iodoacetate and iodoacetamide were without effect. 6. Ca2+, Mg2+, and Zn2+ or EDTA did not influence the enzyme activity.

DNA-Profiling of stains in criminal cases: analysis of measurement errors and band-shift. Discussion of match criteria

DNA-profiling was performed on approximately 600 stains (blood, semen/vaginal secretion, tissue samples and saliva) deriving from criminal investigations. The restriction enzyme was HinfI, and the VNTR (variable number of tandem repeats) probes were MS1, MS31, MS43a and YNH24. DNA-profiles were obtained from 60% of the stains, and matches were seen for 65% of the profiles. The measurement errors and the differences between corresponding fragment lengths of blood and stain profiles were analysed statistically. Distinct band-shifts were observed for approximately 65% of the profiles. For 50% of the profiles, the fragments derived from the stain fragments migrated faster than those from the blood sample, and for 15% of the profiles the stain fragments migrated slower. The difference between the migration distance of the stain and the blood fragments of a given pair of profiles increased with increasing migration distance, i.e. with decreasing fragment length. After correction for this slope the measurement errors were independent of the fragment length, and of the same order of size as for duplicate determinations of fragments from blood samples. The differences between the fragment lengths of corresponding profiles were highly correlated (rho = 0.8). Based on the statistical analysis, different match criteria are discussed and an ellipsoid accept-area is suggested.