Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Studies on avian erythrocyte metabolism: IX. Relationship of changing organic phosphate composition to whole blood oxygen affinity during development of the ostrich (Struthio camelus camelus)

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in erythrocytes (RBC) of the 37-day ostrich embryo at a concentration of 3.7 μmole/ml RBC, representing 23.5% of the cell phosphate. (2) Inositol tetraphosphate (inositol-P₄) is absent in red cells of the 37-day embryo, appears in the RBC about 63 days after hatch, and thereafter gradually accumulates to a level of 2.8 μmole/ml RBC in the adult bird, representing 30–35% of the cell phosphate. (3) Inositol pentaphosphate (inositol-P₅) is present in the erythrocytes of the 37-day embryo at a concentration of 0.8 μmole/ml RBC, reaches a peak level of 2.9 μmole/ml RBC by Day 63 after hatch, and thereafter declines to a level of 1.1 μmole/ml RBC in the adult bird. (4) The crossover time where the molar concentrations of inositol-P₅ and inositol-P₄ are equal in the erythrocyte appears to be about 150 days after hatch. (5) The p₅₀ of whole blood from the 37-day ostrich embryo, 5-day ostrich chick, and adult ostrich was 15.4, 31.8, and 24.9 Torr. The p₅₀ of whole blood immediately after hatch correlates best with an abrupt rise in ATP concentration but after 54 days posthatch correlates best with the appearance of increasing concentrations of inositol-P₄ and the decrease in concentrations of ATP and inositol-P₅. (6) The appearance of inositol-P₄ in the cells could be an adaptive mechanism for regulating oxygen supply by switching to a modulator which maintains a higher oxygen affinity than would a predominance of inositol-P₅.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Effect of oleamide on water and sodium ion transport in osmoregulatory organs of vertebrates

In the frog Rana temporaria L., oleamide solution (10 μmole/L) applied to the isolated basal surface of the skin augmented the short-circuit current (SCC) from 59.8 ± 2.5 to 78.2 ± 1.4 μA/cm². When applied to the serous membrane of the urinary bladder, oleamide (1 μmole/L) induced more than a 30-fold increase in osmotic water permeability. The addition of argininevasotocin against the background of oleamide further increased SCC across the skin and osmotic water permeability in the bladder. In Wistar rats, intraperitoneal injection of oleamide (0.1 μmole/L per 100 g of body weight) to non-anesthetized animals after water load reduced diuresis by 22% and increased solute-free water reabsorption and urinary sodium excretion by 31% and 55%, respectively, but did not affect urinary potassium excretion. These findings provide evidence of the similarity between the effects of oleamide and nonapeptide neurohypophyseal hormones on water and ion transport in epithelial cells of osmoregulatory organs in vertebrates.     

Effect of ursodeoxycholate on the bile flow in the rat

The relationship between the bile flow and biliary excretion rate of bile salt was studied by a continuous infusion of ursodeoxycholate and its glycine conjugate in rats. Infusion of glycoursodeoxycholate produced a higher flow rate and higher bile salt concentration than previously reported values for taurocholate. The estimated biliary transport maximum value was 2.21±0.15 μmole/min/100g body weight (mean±SD, N=13). Furthermore, a linear relation was found between the bile flow and bile salt excretion rate for a wide range of bile salt excretion with a slope value of 4.10±0.64 μl/μmole (N=10). These values were close to values previously reported for tauroursodeoxycholate. In contrast, when free ursodeoxycholate was infused, a bile salt excretion rate increased at first to a level of around 1.0 μmole/min/100g body weight with a concomitant bile flow increase, but after one hr, the bile salt excretion dropped sharply and a lower plateau of about half of the initial maximum level was established in the following hr. On the other hand, the bile flow further increased even in the second hr. Consequently, the linear relationship initially observed between the bile flow and bile salt excretion rate became gradually distorted and after one hr even the positive correlation between the two parameters was completely lost. The sharp drop in the bile salt excretion rate was found to be due to the decrease in the taurine conjugate of ursodeoxycholate in the bile. The excretion rate of free ursodeoxycholate remained at a very low level (about 0.1 μmole/min/100g body weight) throughout the experiments. The concentration of ursodeoxycholate in the liver increased sharply in the second hr corresponding to the decrease in the bile salt excretion rate. These results appear to be most easily explained by the thesis that there is a fraction of bile independent of bile salt excretion but dependent on the bile salt concentration in the hepatocyte.     

D2O effects on association behavior and binding properties of phospho- and dephospho-cytoplasmic malic dehydrogenase

Changes in the concentration of three major carbohydrates, e.g., glycogen, trehalose, and cellulose, were determined during differentiation of Dictyostelium discoideum in a stage study. These three carbohydrates consituted 50–63% of the total carbohydrates. Total carbohydrate content per cell aliquot did not change between the aggregation and sorocarp stages of differentiation. The isolation, purification, and characterization of cellulose is described. Cellulose consisted of an alkali-insoluble (alpha) and an alkali-soluble (beta) fraction. Total cellulose accumulated from very low amounts in late pseudoplasmodium cells to about 35% of carbohydrates in mature sorocarps at a rate of 0.07 μmole glucose equiv/min/ml packed cell volume. Purified alkali-insoluble cellulose constituted about 19% of total carbohydrates in mature sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Trehalose constituted 10–11% of the carbohydrates in sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Glycogen, comparing several methods of determination, was rapidly degraded between the culmination and sorocarp stages of differentiation at an average rate of 0.05 μmole glucose equiv/min/ml packed cell volume. The major portion of glycogen was soluble in TCA and constituted 35% of total carbohydrates in aggregated cells and about 11% in mature sorocarps. A minor fraction of glycogen, about 15% of total carbohydrates in aggregated cells, was solubilized by KOH from a TCA precipitate. A mild acidic treatment of solubilized cell constituents increased the glycogen content by 55%, as judged by an enzymatic assay.     

The influence of α-tocopherol and pyritinol on oxidative DNA damage and lipid peroxidation in human lymphocytes

Unscheduled DNA synthesis (UDS) and lipid peroxidation (LPO) were measured in human peripheral lymphocytes from healthy volunteers. These processes were induced by the catalytic system Fe²⁺-sodium ascorbate. The degree of induced LPO was measured spectrophotometrically by the thiobarbituric acid assay. UDS was detected by scintillometric measurement of the incorporation of ³H-thymidine into DNA. The protective action by fat-soluble vitamin E (d,l-α-tocopherol) and the artificial antioxidant pyritinol on UDS and LPO was also investigated.The system Fe²⁺ (2 μmole/1)-sodium ascorbate (30 μmole/1) increased the LPO level in healthy volunteers approximately 2.5 times and the incorporation of ³H-thymidine by 60–70%. α-Tocopherol (0.2 mmole/1) very efficiently suppressed LPO processes (p < 0.01) and the oxidative damage of DNA measured as UDS was also significantly diminished (p < 0.05). Pyritinol had no effect on LPO and UDS under our experimental conditions.     

Rat brain hexokinase: glucose and glucose-6-phosphate binding sites and C-terminal amino acid of the purified enzyme

The binding of glucose and glucose-6-P by pure rat brain hexokinase has been studied by using an ultrafiltration procedure [H. Paulus (1969) Anal. Biochem. 32, 91–100]. Each mole of enzyme (molecular weight 98,000) binds 1 mole of glucose or 1 mole of glucose-6-P. The dissociation constant for the enzyme-glucose complex (0.04 mm) is in excellent agreement with the kinetically determined K_m for this substrate. The dissociation constant for the enzyme-glucose-6-P complex was estimated to be 1.3 μm, substantially lower than values of 7–8 μm obtained by alternative methods. This discrepancy appears to be due to retardation of the passage of the charged glucose-6-P through the ultrafiltration membrane, resulting in an effective increase in the ligand concentration at the membrane surface and thereby a decrease in the apparent dissociation constant. No appreciable retardation of the passage of the uncharged glucose molecule was observed.The binding of glucose-6-P (but not glucose) is prevented in the presence of P_i. This is in accord with a previously suggested model in which binding of P_i is considered to stabilize the enzyme in a conformation having little, if any, affinity for glucose-6-P.Serine was found as a C-terminal amino acid. The method used would not have detected C-terminal proline or tryptophan residues, and thus these cannot be excluded by the present experiments. However, in view of other results indicating that rat brain hexokinase consists of a single polypeptide chain, it seems probable that serine is indeed the only C-terminal amino acid in the molecule.     

Synthesis of Cholesteryl Supports and Phosphoramidite for Automated DNA Synthesis of Triple-Helix Forming Oligonucleotides (Tfos)

Abstract

DMT-Cholesteryl succinylamino solid supports (CPG, loading: 33 μmole/gram and TentaGel loading: 152 μmole/gram) and DMT-cholesteryl phosphoramidite were prepared for use in automated DNA synthesis of cholesteryl modified TFOs in the synthesis scales from 0.2 to 300 μmole. The modified TFOs were found to have a 5 to 50 fold increase in their uptake properties.     

Study of age-dependent structural and functional changes of mitochondria in skeletal muscles and heart of naked mole rats (<Emphasis Type="Italic">Heterocephalus glaber</Emphasis>)

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per μm² of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles–from 0.21 ± 0.01 to 0.29 ± 0.03 μm². Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.     

In vivo preparation of (32P) adenosine 3',5'-cyclic monophosphate

A simple method for the preparation of [³²P]adenosine 3′,5′-cyclic monophosphate (cyclic AMP) is described. A culture of Escherichia coli mutant deficient in cyclic AMP receptor protein is incubated with [³²P]orthophosphate of known specific activities (up to 4000 Ci/mole) for several cell doublings. 10¹² cells of this mutant excrete approximately 1.4 μmoles of cyclic AMP/hr. The extracellular cyclic AMP can be purified by adsorption to charcoal, chromatography on an alumina plate, and paper chromatography.     

Acid release following activation of surf clam (Spisula solidissima) eggs.

Acid release was observed after activation of Spisula eggs with excess KCI. This acid release begins within 20 sec after the activation and continues for 9–15 min. The amount of acid released was 6.8 μmole per milliliter of packed eggs. In Ca-free or Na-free sea water, the acid release is completely inhibited; subsequent addition of the deficient ion leads to acid release and breakdown of germinal vesicles. These results suggest that Spisula eggs release protons after activation in a manner similar to that of sea urchin eggs, and that acid release with concomitant increase in cytoplasmic pH is probably a general event on activation of marine eggs.     

Inhibition of the serum-dependent,amiloride-sensitive sodium transport pathway in human fibroblasts by extracellular divalent cations

Sodium influx in serum-deprived human fibroblasts in a nominally Ca-free, Mg-free medium is significantly higher (17.8 ± 1.9 μmole/g prot/min) than that measured in a medium containing 1.8 mM Ca and 1 mM Mg (10.9 ± 0.7 μmole/g prot/min), and is stimulated dramatically (44.1 ± 6.1 μmole/g prot/min) by the addition of 10% fetal bovine serum (FBS), suggesting that an enhanced influx of Ca ions is not a necessary condition for serum activation of the amiloride-sensitive Na influx pathway. The addition of 2 mM ethylenediaminetetraacetic acid (EDTA) to serum-deprived cells in a low Ca, low Mg medium also results in a dramatic stimulation of Na influx (40.4 ± 3.7 μmole/g prot/min), while the addition of EDTA to cells assayed in a low Ca, low Mg medium in the presence of FBS has no significant effect on Na influx (45.3 ± 4.1 μmole/g prot/min). Thus, the stimulatory effects of FBS and EDTA are not additive. Kinetic analysis in the presence of varying amiloride concentrations indicate that the EDTA-stimulated Na influx occurs via the amiloride-sensitive Na pathway. The activation of Na influx in cells rinsed free of Ca and Mg Can be readily reversed by the addition of Ca or Mg to the assay medium. The Ca concentration required to give 50% inhibition of Na influx is 52 ± 7.6 μM (n = 3) for cells assayed in serum-free medium and 272 ± 29 μM (n = 3) for cells assayed in the presence of 10% FBS. At physiological Ca concentrations (1.8 mM) the Na influx is maximally inhibited by Ca both in the presence and absence of serum. Since Na influx in 1.8 mM Ca medium is 2.5-fold higher in the presence of serum than in its absence, these data suggest that the serum-induced change in the K, for Ca modulation of the amiloride-sensitive Na transport pathway is not sufficient to explain the serum stimulation of Na influx in human fibroblats.     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis

Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-(acryloyl)benzamide) derivatives (1a–1j) using the Horner-Wadsworth-Emmons olefination of diethyl (2-benzamido-2-oxoethyl)phosphonate and appropriate benzaldehydes. A mushroom tyrosinase inhibitory assay showed compounds 1a (36.71 ± 2.14% inhibition) and 1j (25.99 ± 2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and kojic acid (21.56 ± 2.93% inhibition), and docking studies indicated 1a (−6.9 kcal/mole) and 1j (−7.5 kcal/mole) had stronger binding affinities for tyrosinase than kojic acid (−5.7 kcal/mole). At a concentration of 25 μM, 1a and 1j were nontoxic in B16F10 melanoma cells and exhibited stronger tyrosinase inhibition (59.70% and 76.77%, respectively) than kojic acid (50.30% inhibition) or arbutin (41.78% inhibition at 400 μM). Similarly, in B16F10 melanoma cells, compounds 1a and 1j at 25 μM decreased total melanin content by 47.97% and 61.77%, respectively (kojic acid; 38.98%). Similarities between inhibitions of tyrosinase activity and melanin contents suggested the anti-melanogenic effects of 1a and 1j were due to tyrosinase inhibition. The excellent DPPH scavenging activity of 1j suggests it might enhance in vivo effect on melanin contents. The study suggests compound 1j offers a potential starting point for the development of safe, potent tyrosinase inhibitors.     

Bacillus subtilis aminopeptidase: purification, characterization and some enzymatic properties.

The extracellular aminopeptidase from Bacillus subtilis was purified 300-fold by a simple procedure which gave a high recovery of enzyme. The native enzyme was shown to be a monomer of molecular weight 46,500 and to contain 1 g-atom of Zn²⁺ per mole of protein. Amino acid analyses demonstrated the protein to be rich in acidic residues and Lys, to possess about 3 residues of Met, and to be devoid of Cys. When activated with 5 mm Co(NO₃)₂ for 90 min the activity of the native enzyme was increased; the amount of activation depended on the identity of the substrate. Cobalt activation involved the reversible binding of 1 g-atom of Co²⁺ per mole of protein, without displacing the native Zn²⁺; K_Co was 1.25 mm. Zinc ions competed with Co²⁺ during activation, a process characterized by a _KZn of 28 μm. Ions other than Co²⁺ did not appreciably activate the enzyme.     

Characterization of gastric mucosal membranes. Composition of gastric cell membranes and polypeptide fractionation using ionic and nonionic detergents

Lipid, carbohydrate, amino acid, and polypeptide composition of 2 fractions of cell membranes obtained by sucrose-Ficoll zonal density gradient fractionation of gastric mucosal homogenates has been studied. The membranes with density = 1.04 contain 0.7 μmole lipid P and 0.54 μmole cholesterol/mg protein, while the membranes of density 1.10 contain only 0.25 μmole lipid P and 0.28 μmole cholesterol/mg protein. Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids. Free fatty acids were present. Carbohydrates were most abundant in the proteins of the Peak II membranes (density = 1.10). One polypeptide band was dominant on sodium dodecyl sulfate, Triton X-100, and Brij 36T gels, and the adenosine triphosphatase activity in Brij 36T gels was found to have a relative mobility of 0.14 and 0.19 in the two fractions.     

Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids

Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichlorofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol increases the oxidation of leuco-dichlorofluorescein 5-fold, probably by acting as a cyclic intermediate in the oxidation. Tetramethyl-p-phenylenediamine is also oxidized as cosubstrate. Its oxidation is not influenced by phenol. A stoichiometry of close to one mole of tetramethyl-p-phenylenediamine or leuco-dichlorofluorescein consumed per mole of arachidonic acid was found in the initial phase of the reaction. In the presence of phenol + leuco-dichlorofluorescein, the oxidation rate of arachidonic acid is about 40% lower than with phenol alone as cosubstrate. Since dichlorofluorescein has a molar extinction coefficient of 91 · 10³ at 502 nm, the oxidation of less than 1 μM leuco-dichlorofluorescein can be detected spectrophotometrically. The rate of extinction change with leuco-dichlorofluorescein (at 502 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediamine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, γ-linolenic acid, eicosapentaenoic acid, are substrates for prostaglandin H synthase with decreasing reaction rates in the mentioned order. The same order of reaction rates were found when oxygen consumption was measured. The assay also shows that docosahexaenoic acid is substrate for the enzyme. The reaction rate of the enzyme evidently is decreased both by a n − 3 double bond and by deviation from a 20 carbon chain length of the fatty acid substrate.     

Free amino acids in fluids and tissues of Mytilus galloprovincialis in relation to the environment. Their behaviour as an index of normality of metabolism

• 1.1. A population of Mytilus galloprovincialis has been sampled at 6 different stations of the Bay of Naples (Italy) to analyse the behaviour of free amino acids (FAA) of proteic or non-proteic nature, and the relative parameters of environment.
• 2.2. Thirty FAA of proteic and non-proteic nature have been determined in deproteinized tissue and fluids of M. galloprovincialis.
• 3.3. The most frequent FAA were taurine, as representative of the non-proteic amino acid, cystine, alanine and glutamic acid for non-essential proteic amino acids; valine for the essential proteic amino acids; and other ninhydrin-positive constituents.
• 4.4. There were differences in the environment of the colonies of M. galloprovincialis among the several stations and substantial differences in the macromorphological aspect of the animal were found in relation to their source.
• 5.5. The authors conclude that the FAA content is connected essentially to the regulation of the osmotic pressure and that their concentration represents an index of normality of the metabolism (INM): at FAA concentration total values higher than 550 μmole/g of dry tissue corresponds a negative INM with habitat of osmotic stress, while to concentration values between 0 and 550 μmole/g of dry tissue INM is positive or normal with vital habitat for M. galloprovincialis.
• 6.6. The shape of the concentration curves of the single free amino acids was analysed station by station.

     

The determination of allopurinol and oxipurinol in human plasma and urine

A method is described for allopurinol and oxipurinol assay within human plasma and urine in the range expected during therapy. The method is based on high-performance ion-exchange chromatography following an efficient sample purification step using Chelex-100 resin in the Cu²⁺ form. Linear calibration curves are produced for allopurinol over the range 0.05–10 μmole/1 (0.068–1.36 μg/ml) in plasma and 0.005–1 mmole/1 (0.68–136 μg/ml) in urine and for oxipurinol 0.5–100 μmole/1 (0.076–15.2μg/ml) in plasma and 0.1–2 mmole/1 (15.2–304 μg/ml) in urine.