Effects of dietary supplementation of different levels of vitamin B12 on the liver metabolism of laying hens.

BACKGROUND: Vitamin B12 plays an important role in lipid, protein, carbohydrate and nucleic acid metabolism. We investigated the effect of supplementing layers' diets with different vitamin B12 levels on liver metabolism using a liquid chromatography-mass spectrometry-based metabolomic approach to observe and analyse wide-target metabolomics in the liver. RESULTS: We assigned hens to three groups, namely blank control group without vitamin B12 diet (BCG), normal control group with 25 µg kg-1 vitamin B12 (NCG) and vitamin B12 supplement group I with 100 µg kg-1 vitamin (VBSG I). The VBSG I group layers had higher (P < 0.05) vitamin B12 concentration than those from other groups. The egg yolk vitamin B12 concentration increased (P < 0.01) with the increasing vitamin B12 dietary supplemental level. Between the NCG versus BCG, VBSG I versus BCG, and VBSG I versus NCG groups, 11, 20 and 11 metabolites were significantly changed, respectively. The KEGG pathway of vitamin B6 metabolism was significantly impacted in the NCG layers than those from BCG; seven and five pathways were significantly impacted in the VBSG I layers compared with those from BCG and NCG, including pyrimidine metabolism, vitamin B6 metabolism, glycerophospholipid metabolism, etc. CONCLUSION: We concluded that 25 µg kg-1 vitamin B12 supplementation in corn-soybean meal-based layer diet increased the egg yolk vitamin B12 concentration and impacted the vitamin B6 metabolic pathway, and 100 µg kg-1 of it increased the egg yolk and liver vitamin B12 concentrations and impacted vitamin B6 , lipid, nucleic acid and amino acid metabolic pathways. © 2022 Society of Chemical Industry.

Spectrophotometric determination of lamotrigine in pharmaceutical preparations and urine by charge-transfer complexation.

Rapid and sensitive spectrophotometric methods are developed for the determination of lamotrigine (LTG) in pharmaceutical dosage forms and urine samples, based on the formation of the charge-transfer (CT) complexes between LTG as an n-donor and the acceptors: bromocresol green (BCG), bromocresol purple (BCP), and chlorophenol red (CPR). These complexes are studied spectrophotometrically in chloroform solution in order to obtain some information about their stoichiometry and stability of complexation. The analytical parameters and their effects on the extraction of drug from urine samples are investigated. The reactions were extremely rapid at room temperature, and the absorbance values remained unchanged after 24 h for all reactions. Beer's law was obeyed in the concentration ranges 0.15-19.8, 0.15-19.8 and 0.05-34.1 microg x ml(-1) for CPR, BCP and BCG, respectively. The proposed methods were applied successfully for the determination of LTG in pharmaceutical formulations, and human urine samples in the presence of other antiepileptic drugs such as carbamazepine, oxcarbazepine and phenobarbital, with good accuracy and precision.

Antitumor activity of the DNA fraction from Mycobacterium bovis BCG. II. Effects on various syngeneic mouse tumors.

MY-1, a fraction extracted from BCG and composed of 70.0% DNA and 28.0% RNA, was examined for its antitumor activity against 9 different syngeneic mouse tumors. Tumor regression was induced in almost all of the mice bearing any of five kinds of solid tumors by repeated intralesional injections of 100 micrograms MY-1. When cells of some tumors were inoculated intradermally together with MY-1, tumor growth was suppressed, lung metastases were inhibited, and the survival times of mice bearing 1 of 3 leukemic tumors were prolonged. Repeated sc injections with MY-1 in sites remote from tumor cell inoculation or repeated iv injections were more or less effective against three kinds of solid tumors. Mice inoculated with Lewis lung carcinoma cells in a hind footpad and whose legs were amputated 9 days later were given iv or sc injections of MY-1 every other day (8 times in total), resulting in substantial prolongation of survival. No direct cytotoxicity of MY-1 for these tumors could be shown in three kinds of experiments, which indicates that the antitumor mechanism of MY-1 is host mediated. MY-1 was equally effective in mice with or without presensitization with BCG, whereas BCG was much more effective in BCG-sensitized mice. This finding suggests that a delayed-type hypersensitivity reaction elicited by BCG protein is not required for the antitumor activity of MY-1.

Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity.

A fraction extracted from Mycobacterium bovis strain BCG, which was composed of 70.0% DNA, 28.0% RNA, 1.3% protein, 0.20% glucose, and 0.1% lipid and of no detectable amounts of cell wall components such as alpha, epsilon-diaminopimelic acid and hexosamine, was found to possess strong antitumor activity. Repeated intralesional injection of this fraction, designated MY-1, without attachment to oil or a single intralesional injection of MY-1 emulsified in mineral oil caused the IMC carcinoma of CDF1 mice and line 10 tumor of strain 2 guinea pigs to regress and/or prevented metastasis very effectively. MY-1 after digestion with RNase, which contained 97.0% single-stranded DNA with a guanine-cytosine content of 69.8%, was more effective than undigested MY-1 against IMC and line 10 tumor, while MY-1 digested with DNase, which contained 97.0% RNA, had reduced activity, suggesting that the DNA from BCG possessed strong antitumor activity under certain conditions. Details of the extraction procedures and physicochemical characterization of MY-1 were also described.

Experimental research on utilization of bioluminescent technique to substitute the current viability count for BCG vaccine.

The possibility of using the bioluminescent (BL) technique to substitute the traditional viability count of colony forming units (CFU) of BCG vaccine was investigated. The results showed there is a significant dose-dependent correlation between the concentration of standard adenosine triphosphate (ATP) and ATP BL value. The ultrasonic-chloroform method designed by us yielded the best results. The correlation coefficient values (r) of BL of the liquid and lyophilized vaccine were found to be 0.8 155 and 0.8 484 respectively (P less than 0.05). The coefficient of variation (CV) between the BL value of different lots of ATP obtained was 3.2-4.4%, much lower than that of CFU (CV = 10.4-11.2%). The presence of bacterial clumps within the vaccine had great influence on the bacterial ATP value and on the CFU viability count. 2.8 fg of ATP was found in each CFU formed in the vaccine with clumps, while it was only 0.94 fg in the vaccine without clumps, indicating the superiority of the BL method. The BL method has shown a high sensitivity, good reproducibility and simplicity in handling with quick results and high accuracy. Therefore, we consider that the BL method can be used to substitute the CFU method.

[Examination of protein MPB 70 properties in BCG substrains used for the production of Polish antituberculosis vaccines]. / Badanie obecnosci bialka MPB 70 w podszczepie BCG uzywanym do produkcji Polskiej szczepionki przeciwgruzliczej.

The aim of the study was examination of MPB70 protein production by different BCG substrains and testing this protein as reagent in allergenic skin test on BCG vaccinated guinea pigs. Three BCG substrains: Danish 1331 (D), Japanese 172 (J) and Polish BCG Moreau (P) were used for the study. The protein MPB70 received from dr Nagai from Osaka University was used as reference preparation. It has been shown that MPB70 protein of molecular weight about 21,500 Da was present only in BCG Moreau (P) and Japanese (J) substrains. In immunoblotting test monoclonal antibodies reacted additionally with 43,000 Da protein in all tested substrains. We suspect that the detected additional molecule was a dimer of MPB70 protein. The development of skin reactions to MPB70 protein was seen in guinea pigs vaccinated with J and P substrains. The peak of alergy to MPB70 protein was observed 9 weeks after vaccination. In sera of vaccinated animals antibodies against MPB70 protein were detected by ELISA method. The possibility of using MPB70 protein for diagnostic purpose is discussed.

La tuberculosis: un viejo problema aún sin resolver ¿para cuándo una nueva vacuna? / Tuberculosis: an old unresolved problem when will we have a new vaccine?

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Development and validation of an ATP method for rapid estimation of viable units in lyophilised BCG Danish 1331 vaccine.

An assay for quantifying viability in BCG vaccine by determining intracellular ATP content was developed and validated. ATP content was determined by measuring bioluminescence in the presence of luciferin/luciferase. During development and validation the ATP method was compared to the conventional viable count method. A key step to obtain correlation between ATP content and CFU was found to be a period of pre-incubation in a growth medium before ATP determination. During the validation, the robustness, linearity, accuracy, precision, and range were studied. The method validation study showed that the method applied was robust and applicable to determine ATP content in lyophilised BCG for estimating viability in the BCG samples. By comparison with a conventional viable count method, a high correlation between ATP content and the viable count was found; this relationship can be applied in routine quality control to estimate viable count from the ATP content determined in a sample.

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria.

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.

Comparison of Karl Fischer titrimetric and gravimetric methods for determination of moisture content in BCG vaccine.

A model 447 Coulomatic K-F titrimeter was used to determine the water content of seventeen lots of freeze-dried Tice-substrain BCG vaccine. The results were compared with corresponding moisture contents determined by a standard gravimetric method at the time of manufacture. The advantages of the titrimetric method include simplicity, rapidity, convenience, sensitivity, reproducibility and specificity, whereas the gravimetric method is tedious and time-consuming. Although moisture content determined by the K-F titrimeter tended to be higher than that determined by the gravimetric method, the results correlated significantly (r = 0.882, P less than 10(-5]. Alteration of national and international regulations to permit use of the K-F titrimeter is recommended.

A comparison of the rate of oxygen uptake and the adenosine triphosphate content of routine and experimental BCG preparations.

The ATP content and oxygen uptake rate, two parameters of viability of a BCG suspension, are compared. The lack of correlation between measurements made on fresh routine preparations indicates that the ATP measurements is affected by the degree of dispersion of the preparation. Experimental preparations made under standard conditions (constant semi-dry weight and stepwise dispersive grinding) from cultures of the same age as the fresh routine cultures (14 days) had a smaller ATP content, which correlated well with the respiration rate. In experimental preparations from seven-day cultures this correlation was significant. Between days 7 and 14 the ATP content declined much more rapidly than the respiration rate.

Size characterization of Mycobacterium bovis BCG (Bacillus Calmette Guérin) vaccine, Tice substrain.

Reconstituted, lyophilized, attenuated Mycobacterium bovis, Bacillus Calmette Guérin (BCG) vaccine, Tice substrain, was characterized using a Coulter Multisizer and a HIAC/Royco counter. The primary organism has an equivalent spherical diameter approximating 1 micron but the BCG cell suspension is heavily aggregated. The cumulative size distribution of the suspension fits a log-probit plot and this information can be used to determine the total number of particles per ampoule. The instrumental count may be related to the viable count. The state of dispersion was unaffected by mild shear (syringe aspiration or ultrasound) and only slightly affected by the addition of cetylpyridinium chloride or sodium tauroglycolate.

Comparison of photometric and weight estimation of Mycobacterium content in homogenous BCG cultures containing Tween 80.

A comparison of the weight and photometric methods of primary assay of BCG vaccine has been made, using a vaccine prepared in albumin-free medium but containing Tween 80. In the weight method, the bacteria were trapped on a membrane filter; for photometry a Pulfrich Elpho photometer and an instrument of Czech origin were used. The photometric results were the more precise, provided that the measurements were made within two days of completion of growth; after this time the optical density of the suspension began to decrease slowly. The lack of precision of the weighing method is probably due to the small weight of culture deposit (which was almost on the limit of accuracy of the analytical balance) and to difficulties in the manipulation of the ultrafilter.

Induction of antituberculous immunity by polysaccharidic contaminants of crude ribosomal vaccines isolated from Mycobacterium bovis, strain BCG.

Crude ribosomal fractions isolated from the BCG strain of Mycobacterium bovis are capable of inducing antituberculous immunity in CF-1 mice. These crude preparations contain varying amounts of polysaccharides in addition to ribosomal ribonucleic acid and proteins. Whereas these latter two constituents of the crude fractions were found inactive, high levels of antituberculous immunity were induced by a ribosomal sub-fraction significantly enriched with polysaccharides. These results strongly suggest that polysaccharides play an important role in the induction of antituberculous immunity in CF-1 mice immunized with crude ribosomal fractions isolated from the BCG strain and that if ribosomal proteins and/or RNA are of a certain importance, their role would be secondary (complexes providing physical support to polysaccharides, adjuvant, etc.).

Quantitative evaluation of the effectiveness of Connaught freeze-dried BCG vaccine in mice and in guineapigs.

Although the field trials carried out by the Medical Research Council of Great Britain demonstrated that BCG vaccination can confer a substantial degree of immunity against tuberculous infection, it does not follow that BCG substrains other than the one used for those trials will produce equally favourable results. In fact, there is increasing evidence that different BCG strains may differ widely in their protective potency. The experiments described here further confirm these differences. They also show how the determination of the minimum dose of a BCG vaccine capable of delaying the development of tuberculous infection in mice and in guinea-pigs can yield reproducible data that may help to characterize individual BCG strains.The main purpose of these experiments was to determine the protective potency of Connaught freeze-dried BCG vaccine, lot 140, and to compare it with that of three other BCG vaccines. Marked differences were found with respect to the minimum protective dose for mice or guinea-pigs and the degree of immunity and tuberculin allergy produced in guinea-pigs as shown by the dose-response relationships recorded over a wide dosage range. The results suggest that the Connaught vaccine equals or surpasses the other vaccines in effectiveness.Such tests require a reference BCG vaccine of high protective potency for both animals and man.

Cell mass of Mycobacterium bovis BCG estimated by gas chromatography.

The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.

Report on results of a collaborative assay of BCG vaccines organized by the International Associaton of Biological Standardization.

In a collaborative assay initiated by the International Association of Biological Standardization 13 laboratories investigated the properties of 10 freeze-dried BCG vaccines made in different ways by different producers which met the requirements of the World Health Organization. The in vitro tests with respect to the dry weight of the bacteria, the number of culturable particles and the stability during storage at 37 degrees C gave results which showed statistically significant differences between vaccines as well as between laboratories. The use of a reference vaccine in the tests for the number of culturable particles did not improve the precision of the results. These tests are useful as in-process tests to assure the producer that his vaccine will consist of the required amount of bacteria of which a high percentage is living. It was found that all vaccines had a survival of more than 25% after 1 month at 37 degrees C with the exception of two vaccines in rubber-stoppered vials. The two in vivo tests, determining skin reactivity in guinea-pigs and multiplication of BCG in spleens of mice, resulted in nearly the same ranking of the vaccines with respect to their virulence, differing from that of the in vitro tests. In the skin reactivity test the simultaneous injection of both the reference vaccine and a test vaccine into the same animal achieved the elimination of the inter-laboratory variation of the results.

The lipid substances and cytopathic effects in man of BCG strain "Copenhagen" and attenuated vole bacillus strain "Prague".

Both virulent and attenuated acid-fast micro-organisms (including BCG) may provoke in the infected or vaccinated host cytopathic effects characterized by the formation of specific granulation tissue or caseous necrosis. The mycobacterial antigens responsible for these reactions are localized in the lipid substances which are not essential for eliciting immunity to tuberculosis.The lipid contents of 3 mycobacterial strains (BCG-Prague, BCG-Copenhagen, and MP-Prague-the latter an attenuated strain of Myco. microti) were repeatedly analysed during the period 1964-66. The latter 2 strains-of high and low lipid content respectively-were injected intradermally into 2 tuberculin-positive human volunteers and their cytopathic effects studied. The local reaction to MP-Prague was minimal and healed completely after 1 month; severe ulcerous reactions requiring 4 months to heal were observed with BCG-Copenhagen.Electron-microscopic investigation of ultrathin sections showed marked differences in the surface structure of these strains, indicating that they had different ways of disintegrating in vivo.The authors suggest that mycobacterial strains with a low lipid content and established immunogenicity may be more valuable for tuberculosis prophylaxis than BCG or other attenuated mycobacteria.