Development of a HS-SPME-GC-MS method for the analysis of phthalates in glycerin and liquid paraffin: application to safety evaluation of cosmetic packagings.

Migration of molecules from packaging into products is a well-known phenomenon of which the studies in the food and medical industries are regulated in Europe by several legislations. However, for cosmetic packagings, there is no protocol nor specific migration limits available. The objective of this work was to use glycerin and liquid paraffin as cosmetic product simulants to perform a safety assessment on phthalates in 11 plastic packagings used in the cosmetic industry. To study these compounds in the matrices, 2 extraction techniques were compared: liquid-liquid extraction and solid-phase microextraction (SPME). The optimization of the 2 processes of extraction showed that SPME was more adapted to the study. Finally, samples of glycerin and liquid paraffin were analyzed by a SPME-GC-MS method to quantitate 10 regulated phthalates. In glycerin, only DEP was quantitated above the LOQ in 3 packagings, but the concentrations measured were under the set concentration threshold of 0.5 ppm. In liquid paraffin, DEP was quantitated above this concentration threshold. A safety evaluation was so performed by calculating the systemic exposure damage, and the results were finally considered to be safe for consumers.

Long-Term Storage of Cryptosporidium parvum for In Vitro Culture.

The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.

Técnicas anatômicas no ensino da prática de anatomia animal / Anatomical techniques in the animal anatomy practice teaching

A preocupação quanto a conservação de peças anatômicas existe a mais de 5 mil anos, pois o uso de peças cadavéricas são indispensáveis para o ensino, contribuindo com a melhora das habilidades aplicativas, assimilativas e compreensivas da disciplina. Esse trabalho propõe maior utilização das técnicas apresentadas em laboratórios de anatomia, visando abolir o uso do formol como conservante, permitindo um ambiente agradável para a prática da relação ensino-aprendizagem. Para isso foram utilizadas quatro técnicas anatômicas, a criodesidratação, glicerinação, injeção de látex e injeção de vinilite seguido de corrosão, que foram executadas utilizando cães, gatos e órgãos provenientes de doações. Com a criodesidratação e glicerinação os materiais anatômicos ficaram consideravelmente mais leves do que eram quando mantidos em formol, mantendo-se inodoros, diferente do evidenciado na utilização de formol e outros conservantes. As estruturas de estudo das peças ficaram intactas, de fácil visualização e o armazenamento passou a ser feito em caixas fechadas sem qualquer tipo de liquido, mantendo-se assim por até 3 anos. As técnicas de injeção de látex e vinilite seguido de corrosão mostraram total eficiência preenchendo os sistemas injetados, podendo visualizar as ramificações e todo caminho percorrido no sistema circulatório. As quatro técnicas anatômicas estudadas nesse trabalho mostraram ser suficientes para atender as necessidades dos estudantes quanto ao estudo da anatomia, devido à perfeita evidenciação de estruturas externas e internas dos animais.

The concern about preservation of anatomical specimens in existence for over 5000 years, since the use of anatomical parts, are indispensable for teaching and contribute for the improvement of applicative, assimilative skills and understanding of the discipline. This paper proposes better use of techniques in anatomy laboratories, to abolish the use of formaldehyde as preservative, allowing a pleasant environment for the practice of teaching/learning relationship. For this purpose, we used four anatomical techniques, (1) the cryodehydration, (2) glycerin conservation, (3) latex injection and (4) vinylite injection followed corrosion, we executed using dogs, cats and organs from donations. With cryodehydration and glycerin conservation the anatomical materials were considerably lighter than when kept in formaldehyde, odorless unlike in the use of formaldehyde and other preservatives. The structures to be studied were kept intact, easily to view, and the specimens could be stored in closed boxes without any liquid, thus remaining for up to 3 years. The Injection techniques (latex and vinylite followed by corrosion) showed total efficiency to inject filling systems, and allowed a clear view of all ramifications and paths of the circulatory system. The four anatomical techniques studied were shown to be sufficient to meet the needs of students to study anatomy due to the perfect disclosure of external and internal animal structures.

Reference measurement procedure for total glycerides by isotope dilution GC-MS.

BACKGROUND: The CDC's Lipid Standardization Program established the chromotropic acid (CA) reference measurement procedure (RMP) as the accuracy base for standardization and metrological traceability for triglyceride testing. The CA RMP has several disadvantages, including lack of ruggedness. It uses obsolete instrumentation and hazardous reagents. To overcome these problems the CDC developed an isotope dilution GC-MS (ID-GC-MS) RMP for total glycerides in serum. METHODS: We diluted serum samples with Tris-HCl buffer solution and spiked 200-µL aliquots with [(13)C(3)]-glycerol. These samples were incubated and hydrolyzed under basic conditions. The samples were dried, derivatized with acetic anhydride and pyridine, extracted with ethyl acetate, and analyzed by ID-GC-MS. Linearity, imprecision, and accuracy were evaluated by analyzing calibrator solutions, 10 serum pools, and a standard reference material (SRM 1951b). RESULTS: The calibration response was linear for the range of calibrator concentrations examined (0-1.24 mmol/L) with a slope and intercept of 0.717 (95% CI, 0.7123-0.7225) and 0.3122 (95% CI, 0.3096-0.3140), respectively. The limit of detection was 14.8 µmol/L. The mean %CV for the sample set (serum pools and SRM) was 1.2%. The mean %bias from NIST isotope dilution MS values for SRM 1951b was 0.7%. CONCLUSIONS: This ID-GC-MS RMP has the specificity and ruggedness to accurately quantify total glycerides in the serum pools used in the CDC's Lipid Standardization Program and demonstrates sufficiently acceptable agreement with the NIST primary RMP for total glyceride measurement.

Comparison of cryoprotectants in the preservation of Theileria parva sporozoites using an in vitro infectivity assay.

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p < 0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p < 0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.

Factors affecting motion characteristics of frozen-thawed stallion spermatozoa.

Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 x 10(6)/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at > or = 800 x 10(6) spermatozoa/ml (41%/35%, 32%/27%; P < 0.05). Spermatozoa frozen in LAC at a concentration of 20 x 10(6)/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P < 0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM-EYG was the only parameter affected by freezing rate (P < 0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM-EYG and LAC). In SM-EYG, PMOT (38%) and VCL (109 microns/s) were highest when spermatozoa were frozen in 0.5 ml straws (P < 0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24-48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P < 0.05). Experiment 6 evaluated the effects of cooling time to 5 degrees C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM-EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling-time-by-extender interaction (P < 0.05). In SM-EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5 degrees C prior to initiation of freezing than if freezing was initiated at 20 degrees C (P < 0.05). A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23 degrees C with SM-EYG to 400 x 10(6) spermatozoa/ml; 3) cool to 5 degrees C for 2.5 h; 4) package in 0.5 ml straws at 5 degrees C; 5) freeze in liquid nitrogen vapour at -160 degrees C; and 6) thaw for 30 s in 37 degrees C water.

Studies on cryopreservation of Toxoplasma gondii and its antigenicity in mice.

Two strains (RH and GC, the latter of which is a Taiwan isolate of porcine origin) of Toxoplasma gondii were kept at -20 degrees C, -60 degrees C, and in liquid nitrogen (-196 degrees C) to follow the time course change in viability and virulence of the parasites by direct count and animal inoculation methods. Changes in antibody titers in some of the mice inoculated with the thawed organisms were assayed by the indirect immunofluorescent antibody test. Viability and virulence of T. gondii were best preserved by storage in liquid nitrogen. Tachyzoites kept in liquid nitrogen for eight years still can lead to the death of the injected mice in 2-3 weeks. Virulence of the tachyzoites could be maintained for eight weeks at most at -20 degrees C and -60 degrees C. Dimethylsulfoxide (DMSO) seemed to be a better cryoprotectant for T. gondii than glycerol, but the DMSO-preserved organisms resulted in fewer tachyzoite-containing peritoneal exudates in inoculated mice than the glycerol-preserved organisms. The local isolate (GC strain) tachyzoites tolerated cryopreservation less well than the RH strain parasites. Low antibody titers (at most 1:64) were produced in mice that survived more than 16 days after inoculation with thawed tachyzoites.

Do enzymatic analyses of serum triglycerides really need blanking for free glycerol?

Distributions of concentrations of free glycerol in clinical plasma obtained for triglyceride assay were compiled to determine the frequency with which increased concentrations of free glycerol posed a potential problem for clinical interpretation of triglyceride results. Clinical histories were studied in patients with increased concentrations of free glycerol to ascertain possible reasons for the increase and to assess the relative clinical importance of glycerol-blank-corrected triglyceride results. Significant increases in free glycerol were very uncommon, usually occurring in patients receiving glycerol-containing hyperalimentation fluids, those receiving heparin (which causes both in vivo and in vitro increases in free glycerol), or those critically ill. Free glycerol was never increased significantly in a large outpatient population. Monitoring lipid metabolism in critically ill patients, or measuring true triglyceride concentrations in patients receiving glycerol-containing fluids, may represent rare exceptions for which glycerol-blank correction is necessary for accurate clinical diagnosis and management. We conclude that there is insufficient justification for the routine expenditure of extra time and reagents to correct most analytical enzymatic triglyceride methods for free glycerol.

A simplified method for quality control of deglycerolized erythrocytes.

Quality control to detect inadequately deglycerolized red blood cells can be easily and inexpensively accomplished by suspending the deglycerolized cells in either recipient serum or normal saline in the same way in which the routine crossmatch is performed. In vitro hemolysis is readily and consistently apparent when the residual glycerol exceeds either 2.4% (in saline) or 2.5% (in serum). In contrast to generally held beliefs, the in vivo 24-hour survival of red blood cells with a residual glycerol concentration of up to 2.7% was demonstrated to be 76% of greater, a level which is well above what is usually accepted as adequate.

Inhibition of nuclear DNA-dependent RNA polymerases from mouse ascites tumors and liver by glycerol.

Nuclear DNA-dependent RNA polymerases were isolated from Ehrlich ascites carcinoma, TA3 ascites adenocarcinoma, and mouse liver and tested for inhibition by glycerol. The results confirm the finding of Smith and Duerksen ((1975) Biochem. Biophys. Res. Commun. 67, 916-923) that glycerol may inhibit nuclear RNA polymerase II, but because different grades of glycerol inhibited mouse liver RNA polymerase IIa to different extents, it is suggested that an inhibitory contaminant is present. RNA polymerases IIa and IIb from the two tumors and mouse liver were proportionately inhibited by A.C.S. reagent-grade glycerol at concentrations above 10%. RNA polymerase Ia from liver and the TA3 tumor was not inhibited by any concentration of glycerol tested (2-32.3%), but RNA polymerase Ia from Ehrlich carcinoma was inhibited by glycerol concentrations above 16%.