Interactions of Hydroxyl Radicals with Tris (Hydroxymethyl) Aminomethane and Good's Buffers Containing Hydroxymethyl or Hydroxyethyl Residues Produce Formaldehyde

The production of formaldehyde from tris(hydroxymethyl) aminomethane(Tris) by interaction with hydroxyl radicals(.OH) was studied, since the reaction mixture from the Fenton reaction performed in Tris/HCl buffer was found to be color-developed by colorimetric determination of formaldehyde. The absorption spectrum of chromogens was identical to that of authentic formaldehyde. Color development, which required the presence of Tris, hydrogen peroxide and cupric ions in the Fenton reaction mixture, was inhibited by the addition of hydroxyl radical scavengers such as glucose or hyaluronic acid. These results indicated that formaldehyde was produced when Tris interacted with ·OH. With structures similar to Tris, Good's buffers were also found to produce formaldehyde by interaction with ·OH. Analysis of formaldehyde derived from these buffers may provide a simple and convenient assay for detecting ·OH generation. In evaluating effects of ·OH on the biological system in Tris/HCl buffer or certain Good's Buffers, ·OH loss may be due to interactions of ·OH with these buffers. The formaldehyde produced as a result of such interactions may affect biological systems.     

Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin,Yellowstone National Park

The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with ¹³C-formaldehyde. Isotopic analysis of incubation-derived CO₂ and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.     

The Relationship between Structure and Activity of Taurolin

Taurolin [Bis(1,1-dioxo-perhydro-1,2,4 thiadiazinyl-4)methane] is an antimicrobial compound formed by the condensation of two molecules of taurine with three of formaldehyde. It has been suggested that it releases formaldehyde in contact with bacteria. Evidence from TLC, HPLC and NMR spectroscopy indicates that taurolin is mostly hydrolysed in aqueous solution to release one molecule of formaldehyde and two monomeric molecules (1,1-dioxo-perhydro-1,2,4-thiadiazine and its carbinolamine derivative). A stable equilibrium is established. Antibacterial activity is not entirely due to adsorption of free formaldehyde but also to reaction with a masked (or latent) formaldehyde, as the activity of taurolin is greater than formaldehyde. The monomer is only slightly active by comparison.     

Formaldehyde at low concentration induces protein tau into globular amyloid-like aggregates in vitro and in vivo

Recent studies have shown that neurodegeneration is closely related to misfolding and aggregation of neuronal tau. Our previous results show that neuronal tau aggregates in formaldehyde solution and that aggregated tau induces apoptosis of SH-SY5Y and hippocampal cells. In the present study, based on atomic force microscopy (AFM) observation, we have found that formaldehyde at low concentrations induces tau polymerization whilst acetaldehyde does not. Neuronal tau misfolds and aggregates into globular-like polymers in 0.01-0.1% formaldehyde solutions. Apart from globular-like aggregation, no fibril-like polymerization was observed when the protein was incubated with formaldehyde for 15 days. SDS-PAGE results also exhibit tau polymerizing in the presence of formaldehyde. Under the same experimental conditions, polymerization of bovine serum albumin (BSA) or alpha-synuclein was not markedly detected. Kinetic study shows that tau significantly misfolds and polymerizes in 60 minutes in 0.1% formaldehyde solution. However, presence of 10% methanol prevents protein tau from polymerization. This suggests that formaldehyde polymerization is involved in tau aggregation. Such aggregation process is probably linked to the tau's special "worm-like" structure, which leaves the epsilon-amino groups of Lys and thiol groups of Cys exposed to the exterior. Such a structure can easily bond to formaldehyde molecules in vitro and in vivo. Polymerizing of formaldehyde itself results in aggregation of protein tau. Immunocytochemistry and thioflavin S staining of both endogenous and exogenous tau in the presence of formaldehyde at low concentrations in the cell culture have shown that formaldehyde can induce tau into amyloid-like aggregates in vivo during apoptosis. The significant protein tau aggregation induced by formaldehyde and the severe toxicity of the aggregated tau to neural cells may suggest that toxicity of methanol and formaldehyde ingestion is related to tau misfolding and aggregation.     

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

The known formaldehyde stress response systems involve enzymatic detoxification. Here, the authors show that the formaldehyde sensor efgA plays an important role in the endogenous formaldehyde stress response in Methylorubrum extorquens, halting cell growth in response to elevated levels of formaldehyde, and is found almost exclusively in methylotrophic taxa.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

2012年食用菌“平菇甲醛”事件浅析

对2012年4月媒体报道的青岛平菇中检测发现甲醛所引发的争论进行了剖析。综合分析国内外对食用菌和其他天然食品中甲醛含量的研究结果,以及甲醛自身的理化特性,作者认为食用菌中含有微量甲醛是食用菌自身新陈代谢的产物,从食品安全的角度考虑是安全的。文中介绍了国内外对部分食用菌中甲醛含量的测定、代谢机理和风险评估情况,其中食用菌甲醛的代谢机理值得进一步关注和探讨。     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

核酸(脱)甲基化与内源甲醛及认知损伤

核酸(DNA和RNA)甲基化/脱甲基是表观遗传调控的重要机制.甲醛参与DNA、RNA的甲基化/脱甲基过程,从而影响表观遗传的调节,包括学习记忆等认知功能.然而,甲醛代谢失调将影响核酸的甲基化与脱甲基,使动物的学习记忆能力下降,造成认知损伤.对北京地区604名老人(≥60岁)的调查显示,内源甲醛含量与被试受教育的年限相关,受教育程度越高,内源甲醛含量越低,反之亦然.这些结果表明,内源甲醛在人类学习记忆中扮演重要的角色,"活到老,学到老"可以延缓甲醛代谢失调引起的老年认知损伤.因此,研究内源甲醛代谢与核酸甲基化修饰之间的关系,对探索记忆储存及认知损伤等表观遗传学相关疾病的发生发展机制,具有一定的启示.     

Oxidation of glycerol to formaldehyde by rat liver microsomes

Rat liver microsomes catalyzed the oxidation of glycerol to a Nash-reactive material in a time- and protein-dependent manner. Omission of the glycerol or the microsomes or any of the components of the NADPH-generating system resulted in almost a complete loss of product formation. Apparent Km and Vmax values for glycerol oxidation were about 18 mM and 2.5 nmol formaldehyde per min per mg microsomal protein. Carbon monoxide inhibited glycerol oxidation indicating a requirement for cytochrome P-450. That the Nash-reactive material was formaldehyde was validated by a glutathione-dependent formaldehyde dehydrogenase positive reaction. These studies indicate that glycerol is not inert when utilized with microsomes or reconstituted mixed function oxidase systems, and that the production of formaldehyde from glycerol may interfere with assays of other substrates which generate formaldehyde as product.     

Evaluation of Potential Exposure to Formaldehyde Air Emissions from a Washing Machine Using the IAQX Model

Consumers may be exposed to formaldehyde during the use of liquid laundry detergent containing a preservative. The primary objective of this analysis was to present an approach to predict formaldehyde air emissions from a washing machine and the subsequent vapor concentrations in the laundry room air using the U.S. Environmental Protection Agency's (USEPA's) Simulation Tool Kit for Indoor Air Quality and Inhalation Exposure, referred to as the IAQX model. A second objective was to identify key model input parameters for formaldehyde. This analysis recommends use of the IAQX emission Model 52 because it provided the best estimates by correlating the formaldehyde evaporation to the Henry's law constant and to the overall gas-phase mass transfer coefficient that was based on washing machine experimental results. The mass balance estimated that 99.7% of the initial formaldehyde mass in the washing machine was discharged down the drain with the wash water and the rest of the formaldehyde was emitted to the air from the top loading washing machine and the hot air clothes dryer. The predicted formaldehyde exposures were acceptable and much lower than the USEPA proposed targets for non-cancer effects and cancer risk.     

Levels of formaldehyde and TVOCs and influential factors of 100 underground station environments from 2013 to 2015

In this study, the environmental factors that affect airborne formaldehyde and total volatile organic compounds (TVOCs) were evaluated and the monthly variations in formaldehyde and TVOC levels in underground platforms of subway stations were compared. Formaldehyde level was determined from May 2013 to September 2015 for lines 1 to 4 of Seoul Metro. Samples for formaldehyde and TVOCs were collected at intervals of 30 min during a 60 min period, and then analyzed via high-performance liquid chromatography (HPLC) for formaldehyde and gas chromatography for TVOCs. Formaldehyde levels were correlated with depth of platform, whereas TVOC levels were negatively correlated with depth of platform. There were significant differences in levels of formaldehyde and TVOCs in 2013 and 2015 in the underground platforms. The highest levels of formaldehyde and TVOCs were in July from May to September, respectively (p < 0.05). Formaldehyde and TVOC levels varied greatly, depending on monthly variation and correlated with depth of platform. Therefore, Seoul Metro might need to manage the underground platforms at depth more carefully and further study the reasons behind the relatively high levels of formaldehyde and TVOC ranging from 12 to 22 m.     

Formaldehyde as a pre-treatment for dermal collagen heterografts

The preparation, stability both in vitro and in vivo and resistance to bacterial collagenase of trypsin-purified pig dermal collagen cross-linked with a range of concentrations of formaldehyde in phosphate-buffered saline, was studied using ¹⁴C-labelled formaldehyde as a tracer. Washing in phosphate-buffered saline at 37°C produced rapid loss of formaldehyde over 6 weeks before stability was reached. After 19 weeks washing, 12–20% of the initial radioactivity remained, representing 6, 18 and 35 μmol formaldehyde/g of collagen after 21 days reaction with 0.1, 1 and 5% formaldehyde, respectively. Collagen, incorporating stable-bound formaldehyde arising from reaction with formaldehyde in concentrations of 0.5% or over, was totally resistant to bacterial collagenase.The stabilizing effect of formaldehyde cross-linking was also demonstrated by implants of fibrous pig dermal collagen in rats. After 8 weeks a significant constant amount of formaldehyde was retained in all implants. There was no net loss of mass over a 24 week period when pre-treated with 1% formaldehyde but some loss when pre-treated with 0.1% formaldehyde.     

Purification and characterization of formaldehyde dehydrogenase from rat liver cytosol.

Formaldehyde dehydrogenase was purified to electrophoretic and column chromatographic homogeneity from rat liver cytosolic fraction by a procedure which includes ammonium sulfate precipitation, DEAE-cellulose-, hydroxyapatite-, Mono Q-chromatography, and gel filtration. Its molecular mass was estimated to be 41 kDa by gel filtration and SDS-PAGE, suggesting that it is a monomer. It utilized neither methylglyoxal nor aldehydes except formaldehyde as a substrate. It has been reported that liver class III alcohol dehydrogenase and formaldehyde dehydrogenase are the same enzyme and oxidize formaldehyde and long chain primary alcohols. However, the enzyme examined here did not use n-octanoi as a substrate. The Km values for formaldehyde and NAD+ were 5.09 and 2.34 microM at 25 degrees C, respectively. The amino acid sequences of 10 peptides obtained from the purified enzyme after digestion with either V8 protease or lysyl endopeptidase were determined. From these results, the enzyme was proved to be different from the previously described mammalian formaldehyde dehydrogenase and is the first true formaldehyde dehydrogenase to be isolated from a mammalian source.     

Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes.

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.     

Biodegradation of high concentrations of formaldehyde using Escherichia coli expressing the formaldehyde dismutase gene of Methylobacterium sp. FD1

In the present study, formaldehyde dismutase from Methylobacterium sp. FD1 was partially purified and analyzed by nanoLC–MS/MS; it was then cloned from the genomic DNA of FD1 by PCR. The open reading frame of the formaldehyde dismutase gene of FD1 was estimated to be 1203 bp in length. The molecular weight and pI of formaldehyde dismutase (401 aa), as deduced from the FD1 gene, were calculated at 42,877.32 and 6.56, respectively. NAD(H)-binding residues and zinc-binding residues were found in the amino acid sequence of the deduced formaldehyde dismutase of FD1 by BLAST search. The resting Escherichia coli cells that were transformed with the FD1 formaldehyde dismutase gene degraded high concentrations of formaldehyde and produced formic acid and methanol that were molar equivalents of one-half of the degraded formaldehyde. The lyophilized cells of the recombinant E. coli also degraded high concentrations of formaldehyde.     

Analysis of the Reaction between Formaldehyde and Amide

The reaction between formaldehyde and acetamide which affords a model compound for an amino acid having an amide group was analyzed to investigate the role of formaldehyde as a cross-linking reagent. One of the products was isolated by Sephadex G-10 column chromatography and was identified as N-hydroxymethyl acetamide (FA) by NMR spectrometry and mass spectrometry. Another product, which could not be isolated, was estimated to be N, N-dihydroxymethyl acetamide (F₂A) by kinetic analysis and mass spectrometry. The formation of N, N′-methylene diacetamide was not observed. The mechanism of the reaction between formaldehyde and acetamide was estimated by the kinetic analyses of NMR data, and the rate constants were calculated from the data by the optimization method with a digital computer. On the other hand, formaldehyde cross-linked product was obtained in the reaction of formaldehyde with acetamide and alanine, Its decomposing reaction was analyzed with an NMR spectrometer to study the stability of the formaldehyde cross-linked product. The degradation was dominantly initiated with the release of acetamide.

Consequently, it was estimated that the C–N bond between formaldehyde and amide is so labile that amide-bound formaldehyde does not react further with amides or amines, and that the amide-formaldehyde-amine condensation product is unstable and easily decomposes by releasing the amide.     

The Role of DNase and EDTA on DNA Degradation in Formaldehyde Fixed Tissues

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.     

Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low-Km mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low-Km aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO2 and of formate from [14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30-40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehyde-sensitive (low-Km aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.     

Tissue fixation with phenol-formaldehyde for routine histopathology

Summary The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol—formaldehyde (pH7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol—formaldehyde buffered to pH7.0 and pH5.5 was carried out at an elevated temperature (40°C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol—formaldehyde (pH7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol—formaldehyde (pH7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol—formaldehyde (pH7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol—formaldehyde (pH7.0) and neutral buffered 4% formaldehyde showed similar volume changes.