Linear relationship between the R- and S-enantiomers of β-aminoisobutyric acid in human urine

Urine from untreated patients with various tumours and patients not having tumours has been examined for the excretion of β-aminoisobutyric acid enantiomers. It was shown that the ratio of urinary R- to S-β-aminoisobutyric acid was nearly constant indicating a correlation between the production of both forms. The results are discussed in relation to thymine and valine metabolism, and a metabolic scheme explaining the observed relationship between both forms of β-aminoisobutyric acid is presented.     

High excretion of β-aminoisobutyric acid in patients with ketoacidosis

High concentrations of β-aminoisobutyric acid (BAIBA) were found to be present in the urine from patients with ketoacidosis. The R-form was always the dominating isomer of BAIBA. The finding is discussed, and it is suggested that the mechanism might be a derangement in the degradation of valine.     

Identification of β-aminoisobutyric acid in uremic serum

An unidentified ninhydrin-positive substance found in uremic sera but not found in normal sera was isolated by gel-filtration through Sephadex G-75 followed by high voltage paper electrophoresis (pH 3.5), and identified as β-aminoisobutyric acid using paper chromatography and automated amino acid analyzer.

The quantitative determination of β-aminoisobutyric acid in serum revealed that the level of β-aminoisobutyric acid in uremic sera was much higher than that of normal sera.

Gas chromatographic determination of the enantiomorphs of β-aminoisobutyric acid showed that uremic sera contain R- and S-isomers of the amino acid, but with the R-isomer as the dominating form.     

Heterogeneity of acid phosphatase activity in human polymorphonuclear leukocytes

Evidence is presented indicating the existence of two acid phosphatase activities in human polymorphonuclear leukocytes. There is an acid phosphatase activity, acting preferentially on β-glycerophosphate. This enzyme has a typical lysosomal localization (p = 1.24) and is strongly inhibited by 1.5 mM (+)-tartrate and 15 mM fluoride, being insensitive to 45 mM alloxan and 0.3 M citrate. The second acid phosphatase acts preferentially on phenyl phosphate and is bound to structures of low density (p = 1.18). It is strongly activated by 0.3 M citrate and dl-tartrate, and insensitive to 15 mM fluoride. It is also extremely sensitive to 45 mM alloxan and to thiol-blocking agents, and to denaturation by preincubation under varying conditions.It was not possible to demonstrate any difference in K_m values, pH effect or response to different substances or ions between the β-glycerophospliatase or phenylphosphatase localized in the granular or supernatant fractions. It is concluded that the soluble acid phosphatase activities are similar to that of the granular fraction and may be derived from ruptured lysosomes or solubilization from other subcellular structures.     

Evaluation of the Kodak EKTACHEM clinical chemistry slide for the measurement of bilirubin in newborns

Measurement of neonatal bilirubin using the Jendrassik-Grof method (x) and the EKTACHEM NBIL assay (y) was compared over a 6-month period in a total of 1191 specimens from 483 patients less than 30 days of age. Linear regression analysis of the data yields a slope of 0.937, an intercept of 0.387, an S_y,x of 0.55 and a correlation coefficient of 0.983 for a total of 1032 specimens from patients ? 14 days of age and a slope of 1.090, an intercept of 0.002, an S_y,x of 1.03 and a correlation coefficient of 0.950 for a total of 159 specimens from patients > 14 days of age. The best correlation between EKTACHEM NBIL assay and the reference Jendrassik-Grof method was observed in samples from patients ? 14 days of age. Data from patients older than 14 days showed a higher proportional bias and a lower correlation coefficient between the methods. High performance liquid chromatographic analysis demonstrated that patients > 14 days of age had a higher incidence of elevated β-bilirubin. Linearity extends to 200 mg/l. The NBIL assay provides a rapid, precise micromethod that is less sensitive than the Jendrassik-Grof method to the in vitro photo degradation of bilirubin and is not subject to the interference from hemoglobin and lipids. Because β-bilirubin is not measured by this method, it is only recommended for newborns ? 14 days of age.     

N-Acetyl-L-aspartic acid, N-acetyl-α-l-aspartyl-l-glutamic acid and β-citryl-l-glutamic acid in human urine

$\text{N-}\text{Acetyl-}\text{l}\text{-aspartic}$ acid (NA-Asp), $\text{N-}\text{acetyl}\text{-α-}\text{l}\text{-aspartyl-L-glutamic}$ acid (NA-Asp-Glu) and β-citryl-l-glutamic acid (β-CG), which are known to occur in the brain, have been isolated from human urine. Their identities were proved by comparing them with synthetic NA-Asp, NA-Asp-Glu and β-CG using electrophoretic and Chromatographie methods and by acid hydrolysis.A method was developed for the quantitation of NA-Asp, NA-Asp-Glu and β-CG in human urine. It consists of ion-exchange chromatography followed by gas-chromatographic analysis. The amounts of urinary excretion of NA-Asp, NA-Asp-Glu and ν-CG were 41.2 ± 10.1 (n = 27), 20.8 ± 9.6 (n = 27) and 30.2 ± 13.2 (n = 21) μmol/g creatinine in adult males, and 62.2 ±16.3 (n = 27), 24.0 ±8.2 (n = 27) and 40.5 ± 21.1 (n = 24) μmol/g creatinine in adult females, respectively.     

Enzymatic assay of kynurenine and 3-hydroxykynurenine with Neurospora or Pseudomonas kynureninase

A method is described for the analysis of kynurenine and 3-hydroxykynurenine in biological samples, in particular urine, by use of kynureninase isolated from either Neurospora crassa or Pseudomonas fluorescens. The method is based on the enzymatic conversion of these two metabolites to anthranilic and 3-hydroxyanthranilic acid, respectively, and the selective measurement of anthranilic acid fluorescence at pH 2.7. By this method, it is possible to assay kynurenine and 3-hydroxykynurenine directly in urine samples without prior purification and isolation from other metabolites of tryptophan. The enzymatic reaction is specific for the l-forms of these metabolites and is sensitive enough to detect levels of 1× 10^?7 M.     

A solid-phase method for preparing human DNA from urine for diagnostic purposes

ObjectivesTo develop a simple method using paramagnetic beads for isolation of human DNA from small volumes of urine. The method should be amendable for automation. The purified DNA is intended to be used in downstream diagnostics and screening studies using nucleic acid amplification techniques.Design and methodsUnspecific capture of cells present in urine to magnetic particles, lysis and subsequent binding of the DNA to the same bead surface.ResultsDNA isolated using the method could be used as template for sensitive real-time PCR and end-point PCR using primers targeted to the GAPDH, K-ras, DD3 and p53 genes. Compared to silica spin column-based extraction, the method showed equal or higher DNA yields. The method performed reliably when automated using a liquid handling robot equipped with a magnetic workstation.ConclusionsThe method generates purified DNA free from inhibitors, applicable for sensitive applications such as real-time PCR, genotyping, and for sequence variant analysis. The use of magnetic beads allows for automation, reducing hands-on time and creating a high throughput and reproducible protocol for the purpose of large-scale screening and diagnostics.     

Determination of Bioactives and Antioxidant Activity in Eryngium foetidum L.: A Traditional Culinary and Medicinal Herb

Some of the plant derived bioactives are called as ‘natural antioxidants’ for their role in protecting the cells from injurious effect of reactive oxygen species. The study investigated the concentration of natural bioactives and antioxidant capacity in Eryngium foetidum L. leaves. The antioxidant activity was determined in leaf extracts prepared in five solvents (methanol, acetone, petroleum ether, chloroform and water). The concentration of bioactive compounds (polyphenol, tannin, anthocyanin, flavonoids, carotenoids and ascorbic acid) varied in the extracts prepared with different solvents. The highest recovery of these bioactive compounds was observed with acetone and methanol. The contents of anti-nutritional factors, namely saponin, nitrate, phytate and oxalate content were also estimated in the leaf extracts. HPLC analysis of methanol leaf extract led to detection of several carotenoids (lutein, zeaxanthin, β-cryptoxanthin, β-carotene, chlorophyll-a, chlorophyll-b and pheophytin-b), phenolics (gallic acid, protocatechuic acid, syringic acid, p-coumaric acid, ferulic acid and sinapic acid) and anthroquinones (norlichexanthone, telochistin, secalonic acid D, citreorosein, emodin and parietin). Present study has revealed antioxidant potential of E. foetidum for possible use by food and pharmaceutical industry.     

A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake.     

Isolation and study of human hemopexin (author's transl)

Human hemopexin is a β-glycoprotein which binds hemin, myoglobin and cytochrome c.The isolation method here described involves the use of precipitation with Rivanol, followed by ammonium sulfate precipitation. The final step is recycling chromatography on Sephadex G-100. Hemopexin is obtained in a pure state after four cycles. The sedimentation constant is S₂₀^o= 3.9. The N-terminal amino acid is threonin, and the C-terminal amino acid is histidin. Hemopexin binds hemin through two residues of histidin.     

An improved method for the identification of patients and carriers of Krabbe's disease

The activity of galactosyl ceramide and lactosyl ceramide β-galactosidase was measured in leukocyte and fibroblast homogenates. New incubation conditions for this assay provide a significantly more sensitive method than reported previously. Both reactions were stimulated by optimal concentrations of sodium taurocholate and oleic acid. Citrate-phosphate buffer, pH 4.2, was found to give maximum reaction. Galactosyi ceramide and lactosyl ceramide β-galactosidase activities in leukocytes, cultured skin fibroblasts and cultured amniotic cells from controls and patients with lysosomal disease were compared to carriers and patients with Krabbe's disease. Activity for lactosyl ceramide β-galactosidase was much greater than galactosyl ceramide β-galactosidase activity and may provide a new, more sensitive method for the diagnosis of Krabbe's disease as well as for the identification of suspected carriers. A prenatal diagnosis has been done using this new method.     

beta-Mercaptolactate cysteine disulfiduria in two normal sisters. Isolation and characterization of beta-mercaptolactate cysteine disulfide

β-Mercaptolactate cysteine disulfide (βMLCD) was detected in two mentally normal sisters, 11 and 13 years old. After isolation by ion-exchange chromatography, high-voltage electrophoresis, and adsorption chromatography on Porapak Q, βMLCD was identified by mass spectrometry of its following methyl ester derivatives: O, N-di-trifluoracetyl, O-trifluracetyl-N-dinitrophenyl, O, N-diacetyl, and O-acetyl-N-dinitrophenyl, with acetyl groups 50% perdeuterated, as well as by gas chromatography-mass spectrometry combination of the desulfuration products alanine (as N-trifluotacetyl-methy; ester) and lactic acid (as methyl ester). The isolated βMLCD contained no sulfoxide nor sulfone and exhibited an UV spectrum closely related to cystine. In urine the concentration range of βMLCD was 345–751 μmole/1 (n=5) and 54–133 mg/g creatinine; in plasma a trace of βMLCD could be detected only in one case. Oral administration of L-cysteine or L-methionine elevated the concentration of βMLCD in urine and in plasma. A further disulfide accompanying βMLCD in urine was isolated and identified as thioglycolate cysteine disulfide.     

Dissemination of quinolone low-susceptible Haemophilus influenzae ST422 in Tokyo,Japan

IntroductionHaemophilus influenzae with a reduced susceptibility to quinolones (quinolone low-susceptible H. influenzae) has recently emerged in Japan. In addition, the regional outbreak of the quinolone low-susceptible H. influenzae ST422 clone has been reported. In this study, we isolated this clone from an acute care hospital located in a geographically different area from the previous outbreak and characterised the nature of this clone.MethodsEighty-nine H. influenzae isolated between 2017 and 2019 were tested. The antimicrobial susceptibility was determined by the broth dilution method. The genetic background was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Growth ability and β-lactamase acquisition were evaluated by growth curve analysis and conjugative transfer experiments, respectively.ResultsQuinolone low-susceptible isolates accounted for 4.2% (1/24) in 2018 and 13.9% (5/36) in 2019. Most of the quinolone low-susceptible strains (83.3%) were classified as ST422 and had amino acid substitutions in quinolone resistance-determining regions in both GyrA and ParC. The patients’ backgrounds were highly diverse. In addition, these isolates showed the same PFGE pattern as outbreak strains. The growth of ST422 clone was relatively faster than other clones. Furthermore, ST422 clone was able to acquire β-lactamase from a β-lactamase positive strain by horizontal transfer, becoming highly resistant to β-lactams.ConclusionOur study indicated that the quinolone low-susceptible H. influenzae ST422 clone has been spreading in the community undetected. In addition, this clone has the potential to grow faster and become more resistant through exogenous gene transfer. Therefore, ST422 clone should be monitored attention throughout Japan.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

Evaluation of S1 chromogenic cephalosporin β-lactamase disk assay tested against Gram-positive anaerobes,coagulase-negative staphylococci,Prevotella spp. and Enterococcus spp.

The efficacy of three rapid colorimetric disk assays to detect β-lactamase production in 60 clinical isolates was evaluated. Two chromogenic cephalosporin substrates (S1 and nitrocefin) and an acidimetric test were in complete agreement when tested against Enterococcus spp. (20 strains, not Enterococcus faecalis), Prevotella spp. (10 strains), and Gram-positive anaerobic cocci (10 strains). However, the acidimetric test produced documented false-negative results in detecting the β-lactamases from coagulase-negative staphylococci (two of 20 strains tested). The time required to produce a positive result for the discordant Staphylococcus epidermidis isolate favored S1 compared with nitrocefin. These studies indicate that the acidimetric test was less sensitive than the chromogenic cephalosporin substrates and that nitrocefin and S1 could be used to screen for β-lactamase production in these tested species.     

Practical enzyme immunoassay for prolactin in human serum

We have developed a competitive enzyme immunoasssay for prolactin in human serum. Serum samples were incubated at 4°C for 48 h with anti-prolactin antibodies and prolactin labeled with β-d-galactosidase. The antibody-bound form of labeled prolactin was separated from the unbound form by a method based on the thiol-disulfide interchange reaction. By measuring the enzyme activity, serum prolactin could be determined. Sensitivity of the assay was 2.5 ng/ml, and the assay was as sensitive as radioimmunoassay. There was a good correlation between the values obtained by the enzyme immunoassay and those obtained by a radioimmunoassay (r = 0.98, slope = 1.26, y-intercept = ?12.8 ng/ml).     

Évaluation de l’activité antioxydante et antimicrobienne des feuilles et des sommités fleuries de Marrubium vulgare L.

In the current in vitro study, antioxidant and antimicrobial activities of the organic solvents and aqueous extracts of the leaves and flowered tops of Marrubium vulgare were investigated. Antioxidant activity was evaluated by 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and β-carotene bleaching method, and the antimicrobial activity was evaluated by disc diffusion method. Qualitative analysis of the extracts by thin layer chromatography (TLC) revealed the presence of gallic acid, quercetin and the absence of atropin. Moreover, preliminary identification of the radicalscavenging activity toward DPPH by TLC indicated that the activity is concentrated in the crude methanol extract (MeOHE). Quantification of total phenols by Folin-Ciocalteu method and flavonoids by AlCl₃ method gave higher values with the MeOHE, where the highest value estimated by spectrophotometer measurement: (3.42 ± 0.85 mg GAE/g of extract; 18.0 ± 0.75 mg QE/g of extract). Marrubium vulgare had no activity against tested microorganisms (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853). The β-carotene method focused on the existence of three times: “rest time, generation time, and exhaustion time” with which the MeOHE was characterized by a very high antioxidant activity (63.77%). The quantitative evaluation of the antiradical activity showed that the MeOHE is the most active (IC₅₀ = 1.5 μg/ml); however, quercetin (used as standard) showed approximately equivalent scavenger activity (IC₅₀ = 0.81 μg/ml), indicating the presence of compounds effective in the biochemical composition of the plant which have a high capacity in the reduction of DPPH. These compounds are characterized by a high polarity and are identified by HPLC in the MeOHE of the plant; these compounds are called “phenylpropanoids glycosides”.     

In vitro activity of β-lactamase inhibitors against clinical isolates of Acinetobacter species

Acinetobacter is an important cause of nosocomial infections, and it is often resistant to many antibiotics. In a search for alternative agents, three β-lactamase inhibitors (sulbactam, clavulanate, and tazobactam) and five β-lactam antibiotics (imipenem, ceftazidime, ceftriaxone, cefotaxime, and piperacillin) were tested against 68 unique clinical isolates of Acinetobacter species. Minimum inhibitory concentrations were determined by a broth microdilution method. Using temperature sensitivity testing, we identified 59 strains as Acinetobacter baumannii, one as Acinetobacter haemolyticus, and eight as indeterminate biotype species. We demonstrated 41 of 59 (70%) strains of A. baumannii to be multiply resistant (susceptible only to amikacin and imipenem), whereas all the non-baumannii strains were not. Imipenem was the most active agent among the compounds investigated. All three β-lactamase inhibitors had strong intrinsic activity, with sulbactam being the most active agent among the β-lactamase inhibitors studied.