The term ‘denatured alcohol’ refers to alcohol products adulterated with toxic and/or bad tasting additives (e.g., methanol, benzene, pyridine, castor oil, gasoline, isopropyl alcohol, and acetone), making it unsuitable for human consumption.
The term ‘denatured alcohol’ refers to alcohol products adulterated with toxic and/or bad tasting additives (e.g., methanol, benzene, pyridine, castor oil, gasoline, isopropyl alcohol, and acetone), making it unsuitable for human consumption. The most common additive used is methanol (5–10%), giving rise to the term ‘methylated spirits.’ Denatured alcohol is used as a lower-cost solvent or fuel for home-scale or industrial use, compared with the heavily taxed pure alcohol and alcohol used in beverages.
1.6.6.1 Prohibit the Use of Toxic Substances Such as Methanol in All Types of Surrogate Alcohol That Are Likely to Be Consumed by Humans
The major evidence in this area of intervention exists for the use of methanol as agent to denature alcohol (denatured alcohol is alcohol that is apparently made unfit for human consumption so that it may be exempted from beverage alcohol taxation) (Lachenmeier et al., 2007). In jurisdictions where methanol has been prohibited for this purpose, the morbidity and mortality in relation to methanol-induced surrogate alcohol toxicity was reduced (Anderson et al., 2009). It must be mentioned that there are some substances available (e.g., bittering agents) that may more effectively denature alcohol as methanol, which cannot be tasted in alcohol (Lachenmeier et al., 2007). Nevertheless, methanol poisoning still occurred in 2017 due to illegal sale of methanol-containing surrogate alcohol products, sometimes even mislabeled as “methanol-free” (Hausler et al., 2016; Neufeld et al., 2016).
Similarly, other substances, which would pose acute or chronic toxicity when ingested with alcohol, may be prohibited. Examples are DEP (which has also been used to denature alcohol) or PHMG (which was used as ingredient in disinfectants) (Lachenmeier et al., 2012; Ostapenko et al., 2011; Solodun et al., 2011). Both substances were prohibited in Russia, where a large number of intoxication cases associated with consumption of disinfectant agents as surrogate alcohol were reported (Solodun et al., 2011).
Many unfortunate accidents involving the consumption of methanol have been recorded in the literature. Methanol is often used to denature ethanol for industrial uses, and as its odor is milder and sweeter than ethanol, so its presence in denatured alcohol is difficult to detect. People may accidentally consume methanol while consuming what they believe to be unadulterated ethanol. A report of multiple victims of methanol toxicity in Port Moresby, Papua New Guinea showed that a dose-related response exists with ocular effects ranging from none to blindness. With consumption of higher volumes, vision loss often precedes death. Rarely, significant systemic toxicity may occur via percutaneous or inhalational exposure.
Temporary reactions to systemic methanol exposure include peripapillary edema, optic disc hyperemia, diminished pupillary reactions and central scotomata. Permanent ocular abnormalities include decreased visual acuity, blindness, optic disc pallor, attenuation or sheathing of arterioles, diminished pupillary reaction to light and visual field defects. MRI studies have shown one location of neurological damage from methanol to be in the putamen. Pathologic studies reveal that methanol probably damages mitochondria in the photoreceptors.
One of the most pervasive and potentially dangerous areas of fraud is the illicit trade in alcohol in which it has been estimated that nearly 26% of all alcohol consumed globally is counterfeit or from illegal production.16 The problem of illicit alcohol is ubiquitous and exists in both developing and developed countries alike. It is often linked to organized crime and often has the worst attributes of counterfeiting due to the impact on human health caused by the use of cheap sources of inadequately distilled spirits, or industrial or denatured ethanol. Formulations, of denatured alcohol, which should be used as a solvent or fuel, intentionally contain ‘contaminants’ such as pyridine and/or methanol to prevent its recreational use. At the same time, these additives make denatured alcohol toxic and potentially fatal if consumed in large quantities.17 Denatonium benzoate is often added as an aversive agent (bitterant known as Bittrex) to dissuade human consumption of even the smallest quantities. In addition, these additives are designed to be difficult to remove, however sodium hypochlorite, in the form of household bleach, is used to remove the Bittrex.18 This process does not remove methanol, which is acutely toxic and has led to many serious neurological defects, kidney failure, blindness, and fatalities. The World Health Organization has tracked methanol poisonings involving illicit alcohol in Cambodia, the Czech Republic, Ecuador, Estonia, India, Indonesia, Kenya, Libya, Nicaragua, Norway, Pakistan, Turkey, and Uganda. Each of those instances involved 20–800 victims.19
Krebs-2 ascites tumor cells are maintained using passages in the peritoneal cavity of mice (Villa-Komaroff et al., 1974).
1.
Select a mouse that had been injected 8 days previously and exhibits abdominal swelling. Euthanize the mouse by cervical dislocation or another approved method (sedating the animal by CO2 prior to sacrificing is recommended). Pin the limbs to a Styrofoam support exposing the stomach. Saturate the skin with 70% alcohol (denatured).
2.
Withdraw ascites fluid using a 10-ml syringe and a needle that allows for good flow (18 gauge).
3.
Change to a 26-gauge needle and inject 0.25 ml per mouse in the peritoneal cavity. After 8 days the mice will develop a large quantity (typically 4 to 8 ml) of ascites fluid containing ~108 cells/ml, and their abdomens will be swollen.
4.
If cell extract isolation is intended, conduct one or two additional cell passages, so that 10 to 15 mice with well-developed tumors are obtained.
5.
If cells are being frozen, add an equal volume of ice-cold EBBS containing 20% DMSO to the ascites fluid. Deliver 1 ml of cell suspension (approx 5 × 107 cells) into each freezing vial. Place vials into Nalgene Cryofreezer. Incubate overnight at −80°, then transfer the vials into a liquid nitrogen storage tank.
6.
To expand cells from a frozen stock, you should have one or two mice ready for injection. Thaw frozen cells by briefly placing the vial into a 20° water bath. Disinfect the outside of the vial with 70% alcohol (denatured). Once thawed, the cell suspension should be immediately used for injection (0.5 ml per mouse). Timing is important, since even brief storage of ascites at room temperature could result in its clotting and cell death.
Unrecorded alcohol may be any alcohol discussed previously (ie, wine, beer, and spirits), with the difference that its production was not officially registered in the jurisdiction where the alcohol was consumed, either by illegal production, by home production (which may be licit in some legislations), or by cross-border trade. Besides these groups of beverage-type unrecorded alcohol, other forms of alcohol may be illicitly brought into the food chain (so-called surrogate alcohols), which were not produced as intended for human consumption (such as cosmetics alcohol, medicinal alcohol, disinfectant alcohol, denatured alcohol, synthetic alcohol, or other forms of industrial alcohol). For more details on definition of unrecorded alcohol, see Lachenmeier et al. (2007a, 2009b).
Besides targeted analysis for toxic constituents and adulterants (see review in Lachenmeier et al., 2011), the major question in the authentication of unrecorded alcohol is therefore the differentiation from recorded types of alcohol. This may be difficult when normal alcohol producers may illegally sell part of their alcohol without paying taxes, or when alcohol is smuggled from a low-tax state to a high-tax state (diversion fraud, which was described as common in the EU (WHO Regional Office for Europe, 2009)). Such types of alcohol may be absolutely identical to the conventional, taxed products and therefore not distinguishable by any of the previously mentioned methodologies. Such cases can only be resolved by other means (eg, different labeling or bottle types, or missing or forged tax labels, where required). The computerization of the movement and surveillance system of excisable products and improved enforcement was suggested in the EU as an effective tool against these forms of unrecorded alcohol (Commission of the European Communities, 2004; WHO Regional Office for Europe, 2009).
In cases where no prior information about composition of the unrecorded alcohol is available, a nontargeted analysis may be most preferable. Currently, only NMR has been suggested for this purpose to detect hazardous samples in a collection of unrecorded alcohol samples (Monakhova et al., 2012). This method was able to select samples that contained high concentrations of contaminants or denaturants, such as methanol, ethyl carbamate, diethyl phthalate, or polyhexamethyleneguanidine (Fig. 21.4).
Figure 21.4. Nontargeted analysis of unrecorded alcohol to detect hazardous samples. A scatter plot of the principal component analysis (PCA) scores (10–0.25 ppm) of 1H NMR spectra of 304 samples of international unrecorded alcohols is shown. The outlying samples include denatured and medicinal alcohols from Russia and two samples with extreme concentrations of ethyl carbamate from Romania.
Reprinted from Monakhova, Y.B., Kuballa, T., Lachenmeier, D.W., 2012. Nontargeted NMR analysis to rapidly detect hazardous substances in alcoholic beverages. Applied Magnetic Resonance 42, 343–352 with kind permission from Springer Science and Business Media.
The differentiation of surrogate alcohol from beverage alcohol is sometimes possible, especially when denatured alcohol is sold in pure form or admixed to beverage alcohol. Several methods were suggested in the literature to detect denaturants including diethyl phthalate typically with GC/MS (Leitz et al., 2009; Monakhova et al., 2011a). Denatonium benzoate (bitrex) can be detected using a portable Raman spectrometer (Kwiatkowski et al., 2014) or by liquid chromatography (Daunoravicius et al., 2006; Faulkner and DeMontigny, 1995; Henderson et al., 1998; Kovar and Loyer, 1984), GC/MS (Ng et al., 1998), or UV/VIS spectrophotometry (Bucci et al., 2006). The compound polyhexamethyleneguanidine, which was observed in some surrogate alcohols based on disinfectant solutions, may be detected by spectrophotometry (Solodun et al., 2011).
Three different conformations of DNA exist in nature: A-, B-, and Z-forms (Figure 21.13). The DNA structure Watson and Crick described in their 1953 Nature paper is now known as the B-form of DNA, which is the naturally occurring form in cells and under laboratory physiological conditions. In its B-form, the helix makes one complete turn per every 10 bases, and the distance between adjacent base pairs is 0.34 nm. The B-form of DNA may change to the A-form in a high salt or denatured alcohol solution. The A-form of DNA is still a right-handed helix, but one complete turn of the helix in the A-form requires 11 base pairs. The distance between two adjacent base pairs in the A-form is 0.23 nm. Therefore, the A-form is more compact than the B-form, and its bases are tilted 20° relative to the helical axes. The major groove of A-form DNA is narrower, and the minor groove is wider than those of B-form.
Figure 21.13. Three different conformations of DNA structures are shown in this figure.
The Z-form of DNA is quite different from the other two forms, as it is a left-handed helix and base pairs alternate in a dinucleotide repeating structure such as an alternating purine–pyrimidine sequence (5′pCGCGCGCGCG3′OH). This repeating sequence favors the Z-form of DNA. The major and minor grooves of the Z-form show little difference in depth and width. Since CG-rich sequences are usually found in promoter sequences, the biological role of the Z-form of DNA may include regulation of gene expression. Also, it is known that Z-DNA regions may be formed to relieve torsional stress due to DNA loops that form during transcription. The alternating C–G base pairs introduce a zig-zag structure into the DNA backbone; one turn of Z-DNA requires 12 base pairs, and the distance between adjacent base pairs is 0.38 nm. Z-DNA forms in a high salt solution or in a solution of divalent cations.
Voided urine is the specimen of choice for all screening programs and for diagnostic studies of male patients because of the ease of collection and satisfactory results. Catheterized urine is the preferred specimen from female patients. Hydration of patients, collection of the second voiding in the morning, and collection of three successive morning specimens have been recommended by some investigators, but because of the good cellularity that can be uniformly obtained by filtration or cytocentrifugation preparations as shown in Fig. 15.1 and the significant exfoliation from all nonpapillary tumors of the urothelium, examination of a single urine specimen is sufficient. What is important is prompt fixation, which can best be accomplished by collection of 50–100 mL of urine in an equal amount of 50% alcohol. Ethyl alcohol is preferable, but isopropyl or denatured alcohol is acceptable. If the specimen is to be processed by the Saccomanno blending technique, 2% polyethylene glycol (Carbowax) must be added. Urothelial cells remain well preserved for processing for several days, regardless of whether the urine is collected after hydration, during the morning hours, or later during the day. Urine osmolality does not significantly influence cell preservation,14 but a low pH is desirable and ingestion of 1 g of vitamin C at bedtime before examination has been recommended, although not practical for screening purposes.
Bladder Washings
Bladder washings with normal saline or Ringer's solution performed at cystoscopy, if indicated in combination with biopsies or resections, produce a highly cellular specimen that contains more cell clusters and more large, superficial cells than are seen in voided urine. This procedure is recommended whenever cystoscopy is performed and is the specimen that should be used for flow cytometric studies. The washings should also be collected in equal amounts of alcohol for fixation.
Aspirates, Washings, Brushings, and Cell Blocks of Ureters and Renal Pelvis
If lesions in this region are suspected, urine aspirates, washings, and brushings can be obtained by retrograde catheterization.15 These specimens contain larger numbers of superficial and often multinucleated urothelial cells with greater variation in their appearance than bladder urine and must be interpreted conservatively. Cell blocks may be prepared when tissue fragments are present.
Sample Preparation
Smears of Fresh and Fixed Specimens
Direct smears may be prepared after centrifugation of 50 mL of urine for 10 minutes at 1200 rpm. Albuminized or charged slides are recommended for better attachment of cells in direct smears.
Filtration and Cytocentrifugation-Based Preparations
Specimens may be processed using a filtration or a cytocentrifugation protocol. The slides are then stained by the Papanicolaou method.
Cell Blocks
Cell blocks may be prepared when visible tissue fragments are present. Cytopreparatory techniques for urine specimens are described in detail in Chapter 31.
Treatment, e.g. preservation, of flour or dough for baking, e.g. by addition of materials; baking; bakery products; preservation thereof
A23B
Preserving, e.g. by canning, meat, fish, eggs, fruit, vegetables, edible seeds; chemical ripening of fruit or vegetables; the preserved, ripened, or canned products.
A23C
Milk, milk products; milk substitutes; manufacturing; pasteurizing; sterilizing and preserving
A23D
Butter substitutes; edible oils and fats
A23F
Coffee; tea; their substitutes; manufacture, preparation, or infusion thereof A23G
A23J
Protein compositions for foodstuffs
A23K
Feeding-stuffs specially adapted for animals; methods specially adapted for production thereof
A23L
Foods, foodstuffs, or non-alcoholic beverages; their preparation or treatment, e.g. cooking, modification of nutritive qualities, physical treatment
C12C
Brewing of beer
C12F
Recovery of by-products of fermented solutions; denaturing of or denatured, alcohol
C12G
Wine; other alcoholic beverages; preparation thereof
C12H
Pasteurisation, sterilisation, preservation, purification, clarification, ageing of alcoholic beverages or removal of alcohol therefrom.
C12J
Vinegar; its preparation
C13D
Production and purification of sugar juices
C13F
Preparation and processing of raw sugar, sugar, and syrup
C13J
Extraction of sugar from molasses
C13K
Glucose; invert sugar; lactose; maltose; other sugars
1.9.2 Technological sector 25. Agriculture and food processing, machinery and apparatus IPC Description
A01B
Soil working in agriculture or forestry; parts, details, or accessories of agricultural machines or implements, in general
A01C
Planting; sowing; fertilizing
A01D
Harvesting; mowing
A01F
Processing of harvested produce; hay and straw presses; devices for storing agricultural or horticultural produce A01G Culture of vegetables, flowers, fruit, vines, and hops; forestry; watering
A01J
Manufacture of dairy products (devices, apparatus)
A01K
Animal husbandry; care of birds, fishes, insects; fishing; rearing or breeding animals, not otherwise provided for (housing animals, devices, etc.)
A01L
Shoeing of animals
A01M
Catching and trapping of animals; apparatus for the destruction of noxious animals or noxious plants
A21B
Bakers’ ovens; machines or equipment for baking
A21C
Machines or equipment for making or processing dough; handling baked articles made from dough
A22
Butchering; meat treatment; processing poultry or fish
A23N
Machines or apparatus for treating harvested fruit, vegetables in bulk, not provided for elsewhere; apparatus for preparing animal feeding-stuffs
A23P
Foods or foodstuffs; their treatment, not covered by other classes
B02B
Preparing grain for milling; refining granular fruit to commercial products by working the surface
C12L
Pitching and depitching machines; brewing devices; cellar tools
C13C
Cutting mills; shredding knives; pulp presses
C13G
Evaporation apparatus; boiling pans
C13H
Cutting machines for sugar; combined cutting, sorting and packing machines for sugar
Washing of glassware may seem like a mundane, routine laboratory procedure but it is absolutely critical when handling lipids, especially for oxidation analyses. Borosilicate glass found in most beakers, flasks, and test tubes contains metals and also binds and traps metals in surface pores. The metals then provide very active sites to catalyze lipid oxidation and to transform products. Traces of lipids not fully removed during cleaning also provide radicals and secondary products to catalyze oxidation of subsequent samples. Thus, both prevention of oxidation during normal handling and accurate analyses of lipid oxidation require removal of metals, lipids, and other contaminants in all glassware being used.
A simple but effective protocol for routine glassware cleaning uses the following steps: (1) Wash glassware with an acidic, phosphate-free detergent to remove surface residues, rinse three times with tap water followed by three times with double-distilled, deionized water purified to 18-MΩ resistivity, such as that obtained with Milli-Q or Barnstead purification systems; (2) soak glassware overnight in denatured alcohol saturated with potassium hydroxide to saponify lipid traces and hydrolyze other contaminants, including proteins; (3) repeat step 1; (4) soak overnight in 1 N low-metal hydrochloric acid prepared with 18 MΩ water to dissolve metals on glass; and (5) rinse three times with 18-MΩ water and dry upside-down in hot oven to eliminate traces of bound water. Since background metal levels in laboratory reagent acids and alkalis can be very high, all reagents used in these steps should be the highest purity (especially lowest metal content) reasonably available to avoid adding further contamination. Ultrex™ level reagents are desirable but horrendously expensive for glass washing except for highly specialized chemistry.
Special applications and highly sensitive reactions or assays will require soaking glassware for short times in stronger acid solutions such as 6 mol/L hydrochloric acid or aqua regia (1:3 v/v concentrated nitric and hydrochloric acids) after the two cleaning steps described in the previous paragraph. For example, we use aqua regia cleaning before and after lipid reactions with metal catalysts. Other procedures are available for removing trace metals but either have the potential to leave oxidizing components on the glass (chromic, sulfuric, and nitric acids), are toxic (chromic acid) or are not useful for large scale cleaning (McCormick, 2006). Aqua regia solutions are extremely corrosive and must be handled in a hood with great caution to avoid explosions or skin burns. Guidelines for handling strong acids and aqua regia are available online (Princeton University, 2014; University of Illinois, 2015).
Issues with glassware washers. Questions have been raised about use of laboratory glassware washers for lipid oxidation applications. This author has never had the luxury of using a laboratory glassware washer so cannot speak from direct experience. Nevertheless, I will share my concerns about glassware washers for lipid oxidation analyses and individual laboratories can decide for themselves which method is acceptable. First, nearly all laboratory glassware washers are made of stainless steel, which sounds good, but all stainless steel, even the high-purity SS 316, leaches metals in acidic environments. The acidic detergents recommended for applications where proteins and metals must be dissolved increase potential leaching from the stainless steel, and metals dissolved in the wash water then become deposited on the glass. Each washer model must be carefully inspected to assure that water supplies or circulation never contact non–stainless steel metal parts. One copper or galvanized steel connector can be disastrous for lipid oxidation applications. Detergents must be free of phosphates, surfactants, and other components that may remain as residues on the glassware. Washer models claiming water volume reduction recycle small volumes of wash and rinse water. This may be acceptable for general applications, but not when sensitive chemistry is involved because it concentrates contaminants that may adhere to the glass. Rinsing with tap water is totally verboten due to high contamination with metals and other potential oxidation catalysts. Even rinsing with doubly distilled deionized water is questionable since there are many levels of remaining metals in deionized water, depending on generation method and handling. Water purified to 18-MΩ resistivity in systems such as Milli-Q™ or Barnstead™ should be used, but connecting the laboratory cartridge systems to the washer and providing large volumes of this water rapidly would require modifications. Some detergents claim ability to remove metals, proteins, and greases during short wash times that clean by high-pressure water circulation rather than filling the washer and allowing for soaking. This procedure is adequate for most laboratory applications that are not supersensitive to trace contaminants but is untested for lipid oxidation and other redox applications. Overall, this author is skeptical that automatic glassware washers can remove residues and contaminants adequately for lipid oxidation studies, but as noted above, this is without personal experience.
