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This paper presents a novel method of fabricating an enzymatic biosensor for breath analysis using chromatography paper as enzyme supporting layer and a liquid phase layer on top of screen printed carbon electrodes. We evaluated the performance with ethanol vapor being one of the breathing ingredients. The experimental results show that our sensor is able to measure the concentration of ethanol vapor within the range of 50 to 500 ppm. These results suggest the ability of detecting breath ethanol, and it can possibly be applied as a generic vapor biosensor to a wide range of diseases.

Detection of mycotoxins by conventional methods such as ELISA or LC-MS can be expensive and time-consuming. Therefore, paper-based biosensors can be effectively used for on-site analysis, due to their low cost and easy detection procedures. Nevertheless, even when the application of colorimetric methods on paper enhance the simplicity and affordability of multiple determinations, the signal intensity and final readout can be affected by a limited color uniformity. In this work, Ellman’s method for the quantification of aflatoxin B1 was utilized as a model colorimetric assay on paper, in which the test zones were modified with chitosan-immobilized enzyme (AChE). A comparison of the cross-linking effect of genipin on two chitosans of varying molar mass and degree of acetylation, exhibited a greater signal enhancement from the sample with a higher degree of acetylation and molecular weight.

Background: Fingertip prick whole blood collection on chromatography paper is amenable to high-throughput fatty acid (FA) profiling for large clinical and field studies. However, sample storage is problematic because highly unsaturated FAs (HUFAs) in erythrocytes rapidly degrade in samples stored at 220C. Objective: The aim of the current study was to determine the mechanism of HUFA degradation and to develop prevention protocols. Methods: Free fatty acid (FFA) standards and whole blood reference material from a single participant were used to examine sample storage at 220C for up to 90 d in triplicate. Iron chelation with deferoxamine (0‐5000 mg), antioxidant protection with butylated hydroxytoluene (50 mg), cryopreservation with glycerol, and blood drying were examined using whole blood on chromatography strips. Biological replicate blood samples from additional participants (n = 6) with a range of v-3 (n‐3) HUFA concentrations were similarly assessed. Results: FFAs were relatively stable when stored on chromatography strips at 220C. Glycerol treatment prevented HUFA degradation in whole blood reference material for 30 d (45 6 0.4 to 46.8 6 0.1, means 6 SDs) compared to untreated saline controls (45.9 6 1.0 to 6.8 6 0.2). Pretreatment of paper for blood spots with deferoxamine and drying blood before storage slowed, but not entirely prevented, HUFA degradation over 30 d to 22% and 19% below baseline, respectively, compared to 86‐92% in the controls. Protection against HUFA degradation with blood drying and glycerol treatment was confirmed in the biological replicate study and confirmed by prevention of cell lysis. Conclusions: HUFA degradation during storage at 220C appears to be due to hemolysis and subsequent iron-initiated peroxidation. This degradation may be prevented by glycerol, iron chelation, and/or dried blood spotting. A more thorough understanding of methods to prevent degradation during storage is critical with increasing use of FA profiling in large clinical studies. J Nutr doi: 10.3945/jn.114.203679.

Reactions and purifications, including carbon-carbon bond forming reactions, can be carried out on a cellulose support on which the substrates are non-covalently absorbed.

In order to investigate the adduct-forming properties of sterols with very small quantities of these compounds a method has been developed which uses acetylated chromatography paper as the medium in which the adduct can be formed. For the adduct formation of· cholesterol the results appear to be in agreement with those obtained on a larger scale by refluxing a solution of the sterol in the presence of the other component. The method is also suitable for the investigation of the adduct formation of sterols other than cholesterol. The relative humidity of the atmosphere in which the reaction takes place has an important influence on the adduct formation. Two colour reagents for detecting steroids on acetylated paper are described: a solution of benzenesulphonic acid in ether-methanol, and a solution of antimony trichloride in tetrahydrofurane.

To achieve the goal of poliovirus eradication, surveillance of endemic areas is a crucial step in the poliovirus eradication program. Currently, six countries still have endemic poliovirus. We have tested a novel method which uses SDS/EDTA-treated chromatography paper strips to collect and transport poliovirus-containing stool samples. The SDS/EDTA-treated paper strips were soaked with different dilutions of poliovirus-containing feces and stored at different temperatures. After storing the SDS/EDTA paper strips for 5 months at 37 degrees C, poliovirus RNA could be successfully amplified using RT-PCR. Infectivity of wild-type poliovirus type 1, 2, and 3 was lost upon contact with the SDS/EDTA-treated strips. This easy, inexpensive, and biosafe chromatography paper strip method for the collection and transportation of poliovirus samples can be of use in poliovirus surveillance and polio vaccination programs.

Noroviruses are the most common cause of nonbacterial gastroenteritis outbreaks. A novel method was tested that utilizes sodium dodecyl sulfate (SDS)/EDTA-pretreated chromatography paper strips to collect and transport norovirus-containing fresh stool samples. As confirmation, the strips were infected with different dilutions of human norovirus-containing faeces and stored at different temperatures ( −80, −20, 4 ◦ C, room temperature and 37 ◦ C). Reverse transcription (RT)-PCR analysis was carried out after storage of the strips for 1, 7, 14, 60, and 120 days. The presence of amplifiable norovirus RNA on the strips was inversely correlated with the duration of storage and the storage temperature. Norovirus RNA remained detectable for a sufficient amount of time at room temperature or even in warmer climatic conditions to allow transportation of the samples from a field site to a reference laboratory using regular postal mail. To prove that viral infectivity was lost upon contact with the SDS/EDTA-treated strips, feline calicivirus F9 (10 7 TCID50/ml) was loaded onto the strips. After elution and dialysis, no cytopatic effect could be observed upon incubation on Crandell–Reese feline kidney cells. The SDS/EDTA-pretreated chromatography filter paper strips are an inexpensive, biosafe and adequate method to collect, transport and store norovirus samples. © 2004 Elsevier B.V. All rights reserved.

Cellulose chromatography paper provides a novel non-covalent support for synthesis and in-situ purification of multi-dimensional arrays.

The efficiency of several chromatography papers and thin-layer cellulose powders for the separation of amino acids is investigated, using the minimum elution time for a given resolution as the criterion.

Summary The properties of a commercial chromatography paper (Whatman No. 4) were studied with water and nine organic fluids. Imbibition was greater with the more polar fluids and ranged from 22.0 mm3/cm2 for water to 11.9 mm3/cm2 for benzene. Measurements of flow rates under the influence of gravity showed that, the more polar the fluid, the smaller the resistance to flow offered by the paper. The rate of water was 0.23 ml per hour per cm width at 20°.

The adulteration of herbal medicines by dexamethasone or prednisolone is regarded as a serious problem in many communities. Herein, a novel platform for the separation and quantification of both target steroids in herbal medicines based on electrochemical paper-based analytical devices (ePADs) has been created. The ePAD was composed of Whatman SG81 chromatography paper, 3D-printed devices and a commercial screen-printed electrode. Whatman SG81 silica-coated paper was used for the separation of dexamethasone and prednisolone based on the difference in their partition coefficients during the flow of the mobile phase. The optimal mobile phase was composed of 60% ethyl acetate in cyclohexane and required 7 min for separation. The separated steroids on the paper were then quantified by electrochemical detection using differential pulse voltammetry, in which the 3D-printed devices facilitated the measurement. Analytical detection ranges of 10–500 μg mL−1 were obtained for both dexamethasone and prednisolone (r2 = 0.988 and 0.994, respectively). The limits of detection for dexamethasone and prednisolone were 3.59 and 11.98 μg mL−1, respectively, whereas the limits of quantification were 6.00 and 20.02 μg mL−1, respectively. The amounts of both target steroids derived from real herbal medicine samples determined by the proposed method were comparable to those obtained with assays using standard high-performance liquid chromatography. In addition, a simple evaporation step can be used to increase the concentration of the samples before analysis. These ePADs are simple, low-cost, rapid, and very promising for on-site quantitative detection.

In the last decades, there is a growing search for analytical strategies to ensure clinical analysis without the need of laboratory set-up and skilled personnel. Indeed, user-friendly and low-cost devices are highly valued in the era of sustainability for their capability to be applied in low-resource contexts, such as developing countries. To address this issue, herein we report a 96-well paper-based and laboratory setup-free optical platform for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. We used chromatographic paper to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian Blue Nanoparticles (the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relies on the reaction between the enzymatic product thiocholine and Prussian Blue, giving the Prussian White with subsequently Prussian Blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2–15 U/mL range with a detection limit down to 0.8 U/mL. The accuracy was successfully demonstrated by recovery study with spiked serum and by comparing the data with the gold standard method.

This paper describes a paper-based biochemical sensor that realizes redox cycling with close interelectrode distance. Two electrodes, the generator and collector electrodes, can detect steady-state oxidation and reduction currents when suitable potential is held at each electrode. The sensor has two gold plates on both sides of a piece of chromatography paper and defines the interelectrode distance by the thickness of the paper (180 μm) without any micro-fabrication processes. Our proposed sensor geometry has successfully exhibited signatures of redox cycling. As a result, the concentration of ferrocyanide as reversible redox molecules was successfully quantified under the interference by ascorbic acid as a strong irreversible reducing agent. This was possible because the ascorbic acids are completely consumed by the irreversible reaction, while maintaining redox cycling of reversible ferrocyanide. This suggests that a sensor based on the redox cycling method will be suitable for detecting target molecules at low concentration.

Herein, we report three-dimensional paper chromatography (3D-PC) as a micro-chromatographic platform. The method was based on applying the origami microfluidic device for separation, coupled by colorimetric methods for simultaneous determination. The microfluidic device fabrication was a facile printing approach. Two azo food dyes, Tartrazine (E102) and Indigo carmine (E132), were selected as a model analyte, while carbonate-bicarbonate buffer was used as the mobile phase. Our micro-chromatographic device is associated with two big advantages including needing very small volume of mobile phase ( ~12 µL) and ultrafast separation time (~35 s). Under the optimal conditions, the method provided acceptable linear ranges of 0. 0 g L 1–18.0 g L 1 (R2 = 0.997) for E102 and 0.070 g L 1–10.0 g L 1 for E132 and the limits of detection (3σ/slope) were evaluated as 0.620 and 0.060 g L 1, respectively. The proposed method was successfully applied in the separation and quantification of these dyes in commercial food products such as jelly, candy, and four kinds of drink samples without any sample preparation prior to analysis. The mean recovery values for the real sample analysis were in the range of 100.14%–102.38% for E132 and E102 respectively. The inter-device relative standard deviations were in the ranges of 1.5%–11.8%. In total, our chromatographic μPAD is small (1.0 cm × 1.0 cm × 0.5 cm), portable, inexpensive, no need of specialized user, requires low volumes of sample (0.5 μL), and can perform separation using 12 μL of aqueous mobile phase in very short time.

In this work, we present a facile preparation of a paper-based glucose assay for rapid, sensitive, and quantitative measurement of glucose in blood plasma and urine. Two copper phosphorescent complexes [Cu(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline)(2,6-dimethylphenylisocyanide)2][B(C6H3(CF3)2)4] (Cu1) and [Cu(2,9-dimethyl-1,10-phenanthroline)(2,6-dimethylphenylisocyanide)2][B(C6H3(CF3)2)4] (Cu2) and a new silver congener [Ag(P3)CNAg(P3)][B(C6H3(CF3)2)4] (Ag3) (P3 = PPh2C6H4-PPh-C6H4PPh2 [bis(o-diphenylphosphinophenyl)phenylphosphine]) have been synthesized and their oxygen sensing abilities were investigated. The dimetallic phosphine-based Ag3 complex, having a high oxygen sensing ability, was employed as an efficient signal transducer in enzymatic reactions to recognize blood plasma glucose and urine glucose, which provided a wide linear response for a concentration range between 1.0 and 35 mM and a rapid response, with a limit of detection (LOD) of 0.09 mM for glucose. In practical application, this Ag3 paper-based device offers great analytical reliability and accuracy upon monitoring glucose concentrations in blood plasma.

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [(99m)Tc(MIBI)₆]⁺ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [(99m)Tc(MIBI)₆]⁺ since impurities such as (99m)Tc-reduced-hydrolyzed (RH), (99m)TcO(4)(-) and [(99m)Tc(cysteine)₂]⁻ complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.

Traditional methods for detecting lactoperoxidase (LP) are complex and time-consuming, so a test strip was made based on the enzymatic reaction principle to enable quick and convenient detection of LP in raw milk. In this study 0.1 mol/L citric acid (CA)/0.2 mol/L disodium hydrogen phosphate (NaP) buffer solution (pH 5.0), 22 mmol/L 3,3',5,5'-tetramethylbenzidine (TMB), 0.6 mmol/L hydrogen peroxide (H2O2), and 0.5% Tween-20 or 0.3% cetyltri- methyl ammonium bromide (CTAB) were optimal for preparing a quick, sensitive, and accurate LP test strip. The coefficient of variation (CV) of the estimated LP concentrations ranged from 2.47% to 6.72% and the minimum LP concentration detected by the test strip was 1-2 mg/L. Estimates of active LP in sixteen raw milk samples obtained using the test strip or the TMB method showed a good correlation (r=0.9776). So the test strip provides a quick, convenient, and accurate method for detecting the LP concentration of raw milk.

This paper presents a simple and low-cost method for patterning poly(dimethylsiloxane) (PDMS) barriers in porous support such as paper for the construction of flexible microfluidic paper-based analytical devices (μPADs). The fabrication method consisted of contact-printing a solution of PDMS and hexane (10:1.5 w/w) onto chromatographic paper using custom-designed rubber stamps containing the patterns of μPADs. After penetrating the paper (∼30 s), the PDMS is cured to form hydrophobic barriers. Under optimized conditions, hydrophobic barriers and hydrophilic channels with dimensions down to 949±88 μm and 771±90 μm (n=5), respectively, were obtained. This resolution is well-suitable for most applications in analytical chemistry. Chemical compatibility studies revealed that the PDMS barriers were able to contain some organic solvents, including acetonitrile and methanol, and aqueous solutions of some surfactants. This find is particularly interesting given that acetonitrile and methanol are the most used solvents in chromatographic separations, non-aqueous capillary electrophoresis and electroanalysis, as well as aqueous solutions of surfactants are suitable mediums for cell lyses assays. The utility of the technique was evaluated in the fabrication of paper-based electrochemical devices (PEDs) with pencil-drawn electrodes for experiments in static cyclic voltammetry and flow injection analysis (FIA) with amperometric detection, in both aqueous and organic mediums.

The ability to rapidly identify and quantitate, over a wide range of concentrations, anthocyanins in food and therapeutic products is important to ensuring their presence at medicinally significant levels. Sensitive, yet mild, analysis conditions are required given their susceptibility to degradation and transformation. Paper spray ionization has been used to detect and quantify the levels of anthocyanin levels in extracts of fresh and dried elderberries, and elderberry stems, as well as 3 commercially available nutraceutical formulations. The component cyanidin glucosides, including cyanidin-3-sambubioside, cyanidin-3-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-sambubioside-5-glucoside, and the aglycone cyanidin, were readily detected in a range of sources. Quantitation was achieved by establishing a calibration plot from dilutions of a stock solution of cyanidin-3,5-diglucoside containing malvidin-3,5-diglucoside as an internal standard at a fixed concentration. The same standard was used to quantify the anthocyanin content in the fruit and nutraceutical formulations. Wide 5-fold variations in anthocyanin concentration were detected in the nutraceutical formulations from different suppliers ranging from 1050 to 5430 mg/100 g. These concentrations compared with 500 to 2370 mg/100 g measured in the dried stems and fruit, respectively.

Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin

The development of a new tracer based on the cyclic sulfonamides (sultams) was investigated.3-(Methoxy-phenyl-methyl)-1,6-dimethyl-1H benzo[c][1,2] thiazine 2,2-dioxide (benzo-δ-sultam) was synthesized and characterized by elemental analysis, FT-IR spectroscopy and single crystal X-ray structure determination. The prepared cyclic sulfonamide was labeled with non-commercial (62)Zn radioisotope for fast in vivo targeting and Coincidence imaging purposes (radiochemical purity 97 % ITLC, 96 % HPLC, specific activity 20-23 GBq/mmol). In vivo biodistribution of the final complex was investigated in Sprague Dawley(®) rats bearing fibro sarcoma tumor after 2, 4 and 8 h post injection and compared with free Zn(+2) cation.Using instant paper chromatography method, the physicochemical properties of labeled compounds were found sufficiently stable in organic phases, e.g. a human serum, to be reliably used in bioapplications.The complex exhibited a rapid as well as high tumor uptake (tumor to blood ratio 4.38 and tumor to muscle ratio 9.63) resulting in an efficient tumor targeting agent.

Four marine-derived fungal isolates were screened for the production of inulinase enzyme from low cost substrates under solid state fermentation (SSF), one of them identified as Aspergillus terreus showed the highest inulinase activity using artichoke leaves as a solid substrate. Sequential optimization strategy, based on statistical experimental designs was employed to optimize the composition of the medium, including Plackett–Burman and Taguchi's (L9 3^4) orthogonal array designs. Under the optimized conditions, inulinase activity (21.058 U/gds) reached the predicted maximum activity derived from the taguchi methodology, which increased about 4.79-folds the initial production medium. Fructose was produced, as an end product of inulin hydrolysis proving that the enzyme produced was exoinulinase. The marine-derived A. terreus is suggested as a new potential candidate for industrial enzymatic production of fructose from low cost substrate containing inulin as an economic source.

To detect Salmonella more efficiently and isolate strains more easily, a novel and simple detection method that uses an enrichment assay and two chromogenic reactions on a chromatography membrane was developed. Grade 3 chromatography paper is used as functionalized solid phase support (SPS), which contains specially optimized medium. One reaction for screening is based on the sulfate-reducing capacity of Salmonella. Hydrogen sulfide (H2S) generated by Salmonella reacts with ammonium ferric citrate to produce black colored ferrous sulfide. Another reaction is based on Salmonella C8 esterase that is unique for Enterobacteriaceae except Serratia and interacts with 4-methylumbelliferyl caprylate (MUCAP) to produce fluorescent umbelliferone, which is visible under ultraviolet light. A very low detection limit (10(1) CFU ml(-1)) for Salmonella was achieved on the background of 10(5) CFU ml(-1) Escherichia coli. More importantly, testing with more than 1,000 anal samples indicated that our method has a high positive detection rate and is relatively low cost, compared with the traditional culture-based method. It took only 1 day for the preliminary screening and 2 days to efficiently isolate the Salmonella cells, indicating that the new assay is specific, rapid, and simple for Salmonella detection. In contrast to the traditional culture-based method, this method can be easily used to screen and isolate targeted strains with the naked eye. The results of quantitative and comparative experiments showed that the visual detection technique is an efficient alternative method for the screening of Salmonella spp. in many applications of large-sized samples related to public health surveillance.

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

A novel saccharide host containing four boronic acid recognition units on a single DNA duplex terminus was constructed. This construct allowed boronic acid sugar recognition in the context of double stranded DNA to be established while highlighting the benefits of multivalency. Following the solid-phase synthesis of a bis-boronic acid tag, two end-functionalized oligonucleotides with complementary sequences were functionalized through amide ligation. By annealing the boronic acid-DNA conjugates, a tetra-boronic acid DNA duplex was assembled. The saccharide binding ability of this tetra-boronic acid host was revealed through cellulose paper chromatography in the absence and presence of various saccharides. While no appreciable saccharide binding was seen in the case of a bis-boronic DNA conjugate, the increased migration of the tetra-boronic acid host relative to the control sequences in the presence of selected monosaccharides underscored the importance of multivalent effects. We thus identified a requirement for multiple recognition sites in these conjugate systems and expect the results to facilitate future efforts toward applying synthetic recognition systems to the realm of macromolecules.

A reagentless bioactive paper-based solid-phase biosensor was developed for detection of acetylcholinesterase (AChE) inhibitors, including organophosphate pesticides. The assay strip is composed of a paper support (1 x 10 cm), onto which AChE and a chromogenic substrate, indophenyl acetate (IPA), were entrapped using biocompatible sol-gel derived silica inks in two different zones (e.g., sensing and substrate zones). The assay protocol involves first introducing the sample to the sensing zone via lateral flow of a pesticide-containing solution. Following an incubation period, the opposite end of the paper support is placed into distilled deionized water (ddH(2)O) to allow lateral flow in the opposite direction to move paper-bound IPA to the sensing area to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow-to-blue color change. The modified sensor is able to detect pesticides without the use of any external reagents with excellent detection limits (bendiocarb approximately 1 nM; carbaryl approximately 10 nM; paraoxon approximately 1 nM; malathion approximately 10 nM) and rapid response times (approximately 5 min). The sensor strip showed negligible matrix effects in detection of pesticides in spiked milk and apple juice samples. Bioactive paper-based assays on pesticide residues collected from food samples showed good agreement with a conventional mass spectrometric assay method. The bioactive paper assay should, therefore, be suitable for rapid screening of trace levels of organophosphate and carbamate pesticides in environmental and food samples.

Lipopolysaccharides (LPS) were isolated by phenol-water extraction from 34 strains of Veillonella, and examined by paper chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-deoxy-octonate (KDO). Several preparations were also examined for neutral sugars by gas liquid chromatography. The LPS had in common glucosamine, galactosamine, L-glycero-D-manno-heptose glucose and KDO. Most LPS contained galactose, and a few rhamnose. D-glycero-D-manno-heptose was found in LPS from one of the strains. Based on the sugar composition of the LPS, the Veillonella strains could be classified into four chemotypes.

Evidence that extracts of intestinal contents and intestinal mucosa from germfree rats fed a defined diet, inhibit the bactericidal activity of rat serum on E.coli is presented and a partial chemical characterization of the active factor, FIB, is given. FIB was present in an aqueous extract of intestinal mucosa and fœees and was non-dialysable. Extraction with chloroform—methanol (4:1, by vol.) followed by acetone did not reduce the activity of FIB, indicating that lipids were not necessary for effect. The inhibiting factor resisted 100° C for 30 min. FIB was also resistant to proteolysis (pronase) for 8 days, and was recovered in the resulting non-dialysable hydrolysate, whereas the dialysable fraction was inactive. Thiol-blocking agents had no effect on FIB activity, indicating that the factor was not dependent upon intact thiol groups. FIB activity was enhanced by weak acid hydrolysis, causing liberation of sialic acid and fucose. These observations suggest that FIB is either of polysaccharide or glycoprotein nature. A general inhibition of the bactericidal activity of serum exerted by polar high molecular substances was unlikely, since this effect could neither be demonstrated for the plant polysaccharides nor for the bovine glycoprotein fraction tested.

Sera from patients with myasthenia gravis contain antibodies to a citric acid extractable antigen present in heart and skeletal muscle. This antigen is located in the sarcolemmal/subsarcolemmal region of the muscle fibres. The antigen is resistant to pH changes between 2 and 10, but is inactivated by treatment at 56° C for 60 min or at 70° C for 30 min. The antigenic determinant is of protein nature.

Lipopolysaccharide (LPS) has been prepared from four human oral strains of Veillonella by phenol-water extraction of acetone-washed whole cells. LPS from all strains contained 2-keto-3-deoxy-octonate (KDO), heptose, glucose, glucosamine and galactosamine. In addition, galactose was present in one strain, and galactose and ribose in another. The preparations were rich in lipid and KDO.

Sixty-four strains of Neisseria, Moraxella, and Acinetobacter were screened for cellular monosaccharides by gas-liquid chromatography and other chromatographic techniques. The four sugars ribose, glucose, glucosamine, and 2-keto-3-deoxyoctonate (KDO) were detected in all strains. Heptose was detected only in "true neisseriae" (Neisseria gonorrhoeae, N. meningitidis, N. sicca, N. cinerea, N. flavescens, and N. elongata) and in the tentaively named species Moraxella urethralis. Some marked interspecies dissimilarities within groups were revealed. Thus, N. ovis and M. atlantae were characterized by the presence of mannose. Intraspecies differences were also encountered. N. meningitidis strains of serogroups B and C were distinguished from strains of serogroup A by their sialic acid content. This sugar was also detected in two out of three examined strains of M. nonliquefaciens. In Acinetobacter, heterogeneity of monosaccharide patterns was rather pronounced. The results show the applicability of gas chromatographic "monosaccharide" profiles fo whole cells or extracted carbohydrate in bacterial classification and identification, including differentiation at the subspecies level. In addition, such profiles may be useful for monitoring during purification of cellular polysaccharides.

A study was made of the acid-soluble nucleotides present in Acholeplasma (Mycoplasma) laidlawii A during growth. The nucleotide pool of this organism is meagre, both qualitatively and quantitatively. The main nucleotides are uridine mono- and diphosphates, and none of the more complex nucleotide compounds, such as uridine diphosphate (UDP) sugars or sugar peptides, required for cell wall formation, were detected. The nucleotide composition of A. laidlawii A is compared with that of the Streptococcus L-form.

Serum lipids were studied in 14 chronic alcoholic males, aged 30–56 years, after a bout of drinking. Ten male ward assistants, aged 22–58 years, served as controls. 1. No definite differences were found between alcoholics and controls in the level of total lipids and phospholipids in serum. There was a significant increase of serum cholesterol in the alcoholics compared to the controls. 2. Gas liquid, paper and thin-layer chromatographic determinations of the fatty acid composition of serum cholesteryl esters demonstrated significantly lower figures for linoleic acid and significantly higher figures for monounsaturated fatty acids in the alcoholics. 3. Gas liquid chromatographic determinations of the fatty acid composition of phosphoglycerides demonstrated that the decrease of linoleic acid and the increase of monounsaturated fatty acids were even more pronounced than in the cholesteryl esters. 4. Five alcoholics who were restudied two weeks after admission showed a normalization of the fatty acid pattern. The influence of diet and alcohol on the lipid pattern is discussed.

Labelled tetrachloroethane was synthesized by addition of chlorine to 14C-acetylene. The product (0.51 μci/mg) was injected intraperitoneally into female albino mice (dose 0.21–0.32 g/kg) and the elimination of radioactivity was followed for 3 days. Half of the dose (45–61 %) was expired as carbon dioxide and 28 % (range 23–34) of the activity was excreted with the urine. About 16 % of the dose still remained in the animal and less than 4 % was expired unchanged. By paper chromatography and isotope dilution analysis, the urine was found to contain dichloroacetic acid, trichloroacetic acid, trichloroethanol, oxalic acid and small amounts of glyoxylic acid and urea. Half of the urinary activity was not accounted for. On simultaneous injection of benzoate considerable amounts of labelled hippuric acid, about 20 % of the dose, were excreted. It is suggested that the metabolism of tetrachloroethane takes place mainly via a stagewise hydrolytic fission of carbon-chlorine bonds and oxidation to give first dichloroacetic acid and then glyoxylic acid. With part of the tetrachloroethane, a non-enzymic dehydrochlorination occurs with the formation of trichloro-ethylene which was also found in the breath. It is suggested that trichloroethylene is the precursor of the trichloroethanol and trichloroacetic acid found in urine.

Riboflavin-2-14C was administered orally or intraperitoneally to female Wistar rats in order to study the metabolic fate of the vitamin. Almost all the radioactivity was found in the urine, faeces, carcass and expired CO2 within 24 hours. The chemical recovery of riboflavin was lower, indicating partial degradation. Approximately 1% of the radioactivity was recovered as 14CO2. Following oral administration more than half of the radioactivity excreted in the urine was in the form of metabolites. Paper chromatography of urine revealed three unidentified labelled metabolites. After intraperitoneal administration only negligible amounts of non-riboflavin radioactivity were present in the urine. It is suggested from these data that the enzymes responsible for the degradation were saturated at the highest plasma concentrations reached. It is concluded that riboflavin, ingested orally within the dose range of 0.08 to 2.5 mg/rat, is eliminated to equal extents by degradation and by urinary excretion. This also seems to be the case at physiological conditions of vitamin intake.

Chloroacetic-1-14C acid was given intraperitoneally and the elimination of radioactivity was followed for 3 days. 82–88 per cent was found in the urine and about 8 per cent in the expired air. The faeces (contaminated with urine) contained 0.2–3 per cent and 2–3 per cent remained in the animal. Examination of the urine by paper chromatography showed 2 major metabolites, S-carboxymethyl-L-cysteine and thiodiacetic acid, and small amounts of glycolic acid. The amounts were estimated by isotope dilution analysis giving the following results (percentage of urinary activity): Chloroacetic acid 6–22, S-carboxymethyl-L-cysteine 33–43 (free) and 1–6 (conjugated), thiodiacetic acid 33–42, glycolic acid 3–5 and oxalic acid 0.1–0.2. Carboxymethylcysteine was found to be the precursor of thiodiacetic acid but not of carbon dioxide. When glycolate-1-14C was given intraperitoneally, 60–70 per cent of the radioactivity was eliminated as carbon dioxide. Two metabolic pathways are suggested for chloroacetic acid: (1) A major one with an initial formation of S-carboxymethyl glutathione which is converted to S-carboxymethylcysteine, part of which is further metabolized to thiodiacetic acid, and (2) a minor one involving a probably enzymatic hydrolysis of the carbon-chlorine bond with the formation of glycolic acid which is mainly oxidized to carbon dioxide.

The radioactive metabolites excreted in the urine and faeces after administration of riboflavin-214C to rats were investigated. Paper chromatography (n-butanol:acetic acid:water 4:1:5) of urine indicated the presence of five radioactive metabolites. The average Rf-values were 0.10, 0.20, 0.42, 0.53 and 0.63 respectively. Incubation of urine with urease indicated the presence of minute amounts of radioactive urea. The faeces contained two of the metabolites found in the urine. Metabolite Rf 0.63 was identified as lumichrome. The significance of its predominance in the faeces is discussed. Apart from the metabolite Rf 0.53, the excitation spectrum of which is presented, the metabolites did not fluoresce in an aqueous medium. They were more soluble in water than riboflavin. On a Sephadex G-25 column they were eluted more rapidly than riboflavin. Hydrolysis experiments showed that none of the metabolites were conjugates of riboflavin.

1,1,1,2-tetrachloroethane was given subcutaneously to mice (1.2–2.0 g/kg) and the excretion was followed for 3 days. About half of the dose (21–62 %) was expired unchanged. The part metabolized was excreted mainly as trichloro-ethanol (17–49 % of the dose), and to a lesser extent as trichloroacetic acid (1–7 %). The metabolites were identified by paper chromatography, gas chromatography and mass spectrometry and the Fujiwara reaction was used for the quantitative determination of trichloroacetic acid and of trichloroethanol after oxidation to this acid. It is suggested that the initial metabolic reaction is a hydrolytic fission of a carbon-chlorine bond with the formation of trichloroethanol. Trichloroethanol was the precursor of trichloroacetic acid.

The inhibition of the acetaldehyde dehydrogenase activity in rat liver by an unknown dietary factor, present in a commercial standard diet for rats, was studied. The inhibitory factor was located in the calcinated bonemeal fraction of the diet and was identified by infrared spectrophotometry and thin–layer and paper chromatography as cyanamide or a cyanamide derivative. The occurrence and formation of cyanamide in calcinated bonemeal, its effects on the acetaldehyde dehydrogenases and the metabolism of acetaldehyde in rat liver, are discussed.

Labelled 1,1,2-trichloroethane (0.38 μci/mg), prepared by chlorination of ethylene-14C, was injected intraperitoneally (0.1–0.2 g/kg). The elimination of radioactivity was followed for 3 days. 73–87% of the dose was found in the urine, 0.1–2% in the faeces (contaminated by urine), and 16–22% in the expired air. About 3/5 of the expired activity was due to carbon dioxide and the remainder to unchanged trichloroethane. 1–3% of the dose remained in the animal. Examination of the urine by paper chromatography showed 3 major metabolites – chloroacetic acid, S-carboxymethylcysteine and thiodiacetic acid – and small amounts of glycollic acid. The following amounts (per cent of the urinary activity) were estimated by isotope dilution analysis: chloroacetic acid 6–31, S-carboxymethylcysteine 29–46 (free) and 3–10 (conjugated) and thiodiacetic acid 38–42. These results are in good agreement with those previously obtained by the administration of chloroacetic-1-14C acid. It may therefore be suggested that the metabolism of 1,1,2-trichloroethane proceeds mainly via chloroacetic acid. Isotope dilution analysis also revealed small amounts of 2,2-dichloroethanol, 2,2,2-trichloroethanol, oxalic acid and trichloroacetic acid in the urine.

After subcutaneous or intraperitoneal injection of 14C-pentachloro-phenol (14C-PCP) in the mouse (specific activity 1.6 μci/mg, dose 15–37 mg/kg body weight) the distribution of the activity in the body was determined by whole-body autoradiography and by analysis of the individual organs after oxidation to 14CO2. The highest specific activity was found in the gall bladder and its contents, the wall of the stomach fundus, the contents of the gastro-intestinal tract and the liver. This shows that there is both a gastric and a biliary secretion of PCP and/or its metabolites, and excretion in the faeces. Most of the activity (72–83 %) was excreted in the urine in four days, about half of the dose in 24 hours. Only traces (< 0.05 %) were detected in the expired air. Identification and estimation of metabolites were performed by paper chromatography and isotope dilution techniques. All the urinary activity in the first 24 hours was due to PCP and probably tetrachlorohydroquinone. At least PCP was excreted in both the free and the conjugated form.

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsquently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.

Hydroxylysine was detected in hydrolysates of the inner and outer membranes of the shell of the hen's egg by ion-exchange and paper chromatography. N-ɛ-DNP-hydroxy-lysine was detected by paper chromatography of hydrolysates of dinitrophenylated membranes. Complete amino acid analysis showed hydroxylysine to be the least abundant of the known amino acids in the membranes (1-2 moles/1(fig protein).

A method for the preparation of human STH* was developed on the basis of Wilhelm's technique from 1961 which was simplified, by for example, introducing gel filtration. The purity of the preparation was established by a large number of purity criteria: zone electrophoresis on starch, zone electrophoresis in a density gradient, determination of N- and C-terminal end groups, determination of the N-terminal amino acid sequence, quantitative amino-acid analysis, determination of molecular weight by gel filtration (mol. wt.: 2.2×104), etc. Oxidation of human STH causes polymerization. The alteration of the degree of polymerization of the oxidized product was studied at varying pH levels. The molecular weight of the preparation at the highest degree of polymerization was found to be 45×104.