TiO2hierarchical nano blooming-flower decorated by Pt for formaldehyde detection.

To achieve an ultra-low concentration formaldehyde detection at low temperature, a platinum (Pt) assisted TiO2hierarchical nano blooming-flower sphere material is synthesized through hydrothermal method. SEM and transmission electron microscope characterizations show that the diameter of the nano sphere was around 2µm with dissilient rods of 60 nm in diameter and 1µm in length on the surface. The response (Ra/Rg) achieved form this nanomaterial to HCHO is 1.08 (100 ppb) and 5.82 (5 ppm) at 130 °C without an involvement of any light source or solution. The relationship curve between the responses and concentrations shows regular exponential trend. The verification of sensor stability done by a 3 month reliability test shows no response-degradation. The optimal response and stability is attributed to the massive dissilient rods on the surface of TiO2spheres and the assistance of Pt as a catalyzer disperses to intensify the formation of depletion area on the surface of TiO2. This study provide an attractive and cost effective solution for the detection of HCHO in air at a relatively low temperature.

Circular RNA-CDR1as is involved in lung injury induced by long-term formaldehyde inhalation.

OBJECTIVE: Formaldehyde (FA) is known to induce lung injury, but the underlying molecular mechanism remains largely unclear. CDR1as is an important member of the circular RNAs (circRNAs) family and functions as miRNA sponges with gene-regulatory potential. Our earlier circRNA microarray data showed CDR1as was highly expressed in lung tissue exposed to FA. However, the mechanism of circRNA-CDR1as mediates the FA-exposed lung injury is still unclear. This study aimed to explore the role of CDR1as in lung injury. MATERIALS AND METHODS: In this study, FA was inhaled at doses of 0.5, 2.46, and 5 mg/m3, respectively. After exposure 8 weeks, lung histopathological examination, lung injury score, and IL-1ß in bronchoalveolar lavage fluid (BALF) were determined. The expressions of CDR1as, rno-miR-7b and Atg7 were detected and the potential interaction of circRNA/miRNA/mRNA was predicted by bioinformatics analysis, including drawing circRNA/miRNA/mRNA interaction network, GO and KEGG analysis. RESULTS: Our results indicated FA inhalation upregulated the expression of CDR1as in lung tissues in a dose-dependent manner while the expression of rno-miR-7b decreased and Atg7 increased. Moreover, the alteration of CDR1as was positively correlated with lung injury. DISCUSSION AND CONCLUSIONS: CircRNA/miRNA/mRNA prediction further explained the possible effect mechanisms of CDR1as. These data implicated that CDR1as might be a critical regulator involved in lung injury induced by FA.

Learning and memory impairment of mice caused by gaseous formaldehyde.

In order to study the e of formaldehyde exposure on learning and memory ability of mice. We used Kun Ming (KM) mice to demonstrate the neurotoxic effects of FA, and Balb/c mice to explore the neurobiological mechanism. The Morris water maze (MWM) test showed that the exposure of gaseous formaldehyde could cause spatial learning and memory impairment in mice. H & E staining showed that in the 3.0 mg/m3 formaldehyde exposed group, the arrangement of pyramidal cells in CA1 area of mouse hippocampus was loose and disordered, the cell morphology was swollen and deformed, and the apical dendrites were shortened or even disappeared. Biochemical indicators revealed high doses of FA exposure could cause oxidative damage in brain. Compared with the control group, there were significant differences in the levels of ROS, MDA, GSH and 8-OHDG in the 3.0 mg/m3 group (P < 0.01), also the monoamine neurotransmitters content and the content of TNF-α, IL-1ß and Caspase-3 (P < 0.01). Furthermore, the concentrations of cAMP, cGMP, NO and the activity of NOS in the cerebral cortex, hippocampus and brain stem after high doses of FA exposure were significantly different from those in the control group, indicating that FA exposure could interfere with the transduction of NO/cGMP signaling pathway. The results showed that FA could induce cognitive deficits and this extended investigation found that the toxicity of FA to the mouse nervous system is related to the NO/cGMP and cAMP signaling pathways.

Low concentrations of FA exhibits the Hormesis effect by affecting cell division and the Warburg effect.

Formaldehyde (FA), a ubiquitous indoor environmental pollutant, has been classified as a carcinogen. There are many studies showed that low levels of FA could promote cell proliferation, however, little is known about the signal pathways. To determine the potential molecular mechanisms, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposed to different concentrations of FA. The data showed that FA at 0-125 µM or 0-60 µM promoted the proliferation of K562 cells or 16HBE cells respectively, indicating that FA did have the Hormesis effect. FA at 75 µM (K562 cells) and 40 µM (16HBE cells) significantly promoted cell proliferation, increased intracellular reactive oxygen species (ROS) levels, and decreased glutathione (GSH) content. At the same time, FA treatment induced a marked increase in the key molecules of cell division like CyclinD-cdk4 and E2F1. In addition, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid and lactate dehydrogenase A (LDHA) content in the Warburg effect were increased. Administering Vitamin E (VE), significantly disrupted cell division and disturbed the Warburg effect, effectively indicating the decrease of cell activity. Conclusively, these findings suggested that low concentrations of FA could promote cell proliferation by accelerating cell division process or enhancing the Warburg effect to embody the Hormesis effect.

Formaldehyde inhalation triggers autophagy in rat lung tissues.

Formaldehyde (FA), a ubiquitous environmental contaminant, has long been suspected of causing lung injury. However, the molecular and cellular mechanisms underlying this phenomenon remain elusive. The aim of this study was to elucidate the role of autophagy in lung injury induced by FA inhalation. In this study, lung weight coefficient, interleukin 8 in bronchoalveolar fluid, and histopathological examination were used to evaluate the lung injury. Moreover, electron microscopy, Western blotting for the ratio of LC3-II/LC3-I were used to detect autophagy in lung tissues. Our results indicated that the lung toxicity of FA inhalation is dose dependent. Lung weight coefficient, inflammatory response, and histopathological structure in the 0.5 mg/m3 FA exposure group showed no obvious changes compared with the control. However, exposure to 5 and 10 mg/m3 FA produced lung injury including pulmonary edema, histological changes, and inflammatory responses. Furthermore, the alterations of autophagy correlated with lung injury. Taken together, these data indicate that FA exposure triggers autophagy of alveolar epithelial cells, which might play a pivotal role in lung injury.

Effects of chronic exposure to Formaldehyde on micronucleus rate of bone marrow cells in male mice.

Formaldehyde (FA) is the major volatile organic chemical (VOC) present in indoor air, and a constituent known to be associated with sick building syndrome. In the present study, mice were exposed to different concentrations of FA (0, 1, 10 mg/ m3) through static inhalation for 2 hours per day for 20 weeks. The polychromatic erythrocytes/ normochromatic erythrocyte (PCE/NCE) ratio and the micronucleus rates in bone marrow cells were detected. Data indicated that the PCE/NCE ratio in two FA exposure groups were statistically significant lower than the negative control group (P <0.05), and the micronucleus rate in two FA exposure groups were not significantly higher than the control group (P>0.05). These results suggest that chronic static inhalation of FA can reduce the ratio of PCE/NCE in the mice bone marrow, but the effects to the mice bone micronucleus rate are not sure.

Aß-binding with alcohol dehydrogenase drives Alzheimer's disease pathogenesis: A review.

Although Alzheimer's disease (AD) characterized with senile plaques and neurofibrillary tangles has been found for over 100 years, its molecular mechanisms are ambiguous. More worsely, the developed medicines targeting amyloid-beta (Aß) and/or tau hyperphosphorylation did not approach the clinical expectations in patients with moderate or severe AD until now. This review unveils the role of a vicious cycle between Aß-derived formaldehyde (FA) and FA-induced Aß aggregation in the onset course of AD. Document evidence has shown that Aß can bind with alcohol dehydrogenase (ADH) to form the complex of Aß/ADH (ABAD) and result in the generation of reactive oxygen species (ROS) and aldehydes including malondialdehyde, hydroxynonenal and FA; in turn, ROS-derived H2O2 and FA promotes Aß self-aggregation; subsequently, this vicious cycle accelerates neuron death and AD occurrence. Especially, FA can directly induce neuron death by stimulating ROS generation and tau hyper hyperphosphorylation, and impair memory by inhibiting NMDA-receptor. Recently, some new therapeutical methods including inhibition of ABAD activity by small molecules/synthetic polypeptides, degradation of FA by phototherapy or FA scavengers, have been developed and achieved positive effects in AD transgenic models. Thus, breaking the vicious loop may be promising interventions for halting AD progression.

Formaldehyde toxicity in age-related neurological dementia.

The primordial small gaseous molecules, such as: NO, CO, H2S and formaldehyde (FA) are present in the brains. Whether FA as well as the other molecules participates in brain functions is unclear. Recently, its pathophysiological functions have been investigated. Notably, under physiological conditions, learning activity induces a transient generation of hippocampal FA, which promotes memory formation by enhancing N-methyl-D-aspartate (NMDA)-currents. However, ageing leads to FA accumulation in brain for the dysregulation of FA metabolism; and excessive FA directly impairs memory by inhibiting NMDA-receptor. Especially, in Alzheimer's disease (AD), amyloid-beta (Aß) accelerates FA accumulation by inactivating alcohol dehydrogenase-5; in turn, FA promotes Aß oligomerization, fibrillation and tau hyperphosphorylation. Hence, there is a vicious circle encompassing Aß assembly and FA generation. Even worse, FA induces Aß deposition in the extracellular space (ECS), which blocks the medicines (dissolved in the interstitial fluid) flowing into the damaged neurons in the deep cortex. However, phototherapy destroys Aß deposits in the ECS and restores ISF flow. Coenzyme Q10, which scavenges FA, was shown to ameliorate Aß-induced AD pathological phenotypes, thus suggesting a causative relation between FA toxicity and AD. These findings suggest that the combination of these two methods is a promising strategy for treating AD.

Dual-Targeting into the Mitochondria of Cancer Cells for Ratiometric Investigation of the Dynamic Fluctuation of Sulfur Dioxide and Formaldehyde with Two-Photon Integrated Semiconducting Polymer Dots.

Mitochondrial sulfur dioxide (SO2) and formaldehyde (FA) in cancer cells serve as important signal molecules in mediating multiple physiological and pathological activities. Accurate monitoring of the dynamic fluctuation of SO2 and FA in the mitochondria of cancer cells is important for insight into their relationships and functions in cancer, understanding cancer mechanism, and the role of mitochondrial homeostasis in cancer invasion and metastasis. Herein, a novel integrated two-photon semiconducting polymer dot (BF@Pdots) with dual-targeting (cancer cells and mitochondrial) and dual-emission in green and red regions, which is rationally designed through a four-step engineering strategy by using two newly synthesized functionalized polymers PFNA and FD-PSMA as precursors, has been developed for accurate tracking of the dynamic variation of SO2 and FA in the mitochondria of cancer cells. The sensing mechanism is on the basis of the fluorescence resonance energy transfer (FRET) process in BF@Pdots tuned by the reversible Michael addition reaction between the sensing-groups and SO2 (or FA). The integrated BF@Pdots nanoprobes display excellent performances in the accurate detection of the dynamic fluctuation of SO2 and FA such as precise positioning in the mitochondria of cancer cells, self-calibrating ratiometric, two-photon emission with long wavelength excitation, and fast reversible response. The BF@Pdots nanoprobes are also applied to the ratiometric detection of the dynamic fluctuation of exogenous and endogenous SO2 and FA in the mitochondria of cancer cells for the first time with satisfactory results. Taken together, this work will provide an attractive way to develop versatile integrated Pdots-based fluorescent probes through flexible molecular engineering for applications in accurate imaging of biomolecules in living systems.

Formaldehyde-Crosslinked Nontoxic Aß Monomers to Form Toxic Aß Dimers and Aggregates: Pathogenicity and Therapeutic Perspectives.

Alzheimer's disease (AD) is characterized by the presence of senile plaques in the brain. However, medicines targeting amyloid-beta (Aß) have not achieved the expected clinical effects. This review focuses on the formation mechanism of the Aß dimer (the basic unit of oligomers and fibrils) and its tremendous potential as a drug target. Recently, age-associated formaldehyde and Aß-derived formaldehyde have been found to crosslink the nontoxic Aß monomer to form the toxic dimers, oligomers and fibrils. Particularly, Aß-induced formaldehyde accumulation and formaldehyde-promoted Aß aggregation form a vicious cycle. Subsequently, formaldehyde initiates Aß toxicity in both the early-and late-onset AD. These facts also explain why AD drugs targeting only Aß do not have the desired therapeutic effects. Development of the nanoparticle-based medicines targeting both formaldehyde and Aß dimer is a promising strategy for improving the drug efficacy by penetrating blood-brain barrier and extracellular space into the cortical neurons in AD patients.

Determination of formaldehyde and acetaldehyde levels in poly(ethylene terephthalate) (PET) bottled mineral water using a simple and rapid analytical method.

A simple and rapid analytical method was developed for the determination of formaldehyde (FA) and acetaldehyde (AA) contents in water. FA and AA were derivatized by 2,4-dinitrophenylhydrazine in an LC vial for 20 min at room temperature, about 25 °C, and analyzed using LC-MS/MS. The calibration curve exhibited excellent linearity for FA and AA concentrations of 2-150 ng/mL. Recovery tests using ultra-pure water and commercially available PET-bottled mineral water samples showed good trueness and precision. We determined the FA and AA contents in 105 PET-bottled mineral water samples on the Japanese market using this method. FA was detected in 61% of the samples at levels from 2.6 to 31.4 ng/mL, while AA was detected in 68% at levels from 5.3 to 143.5 ng/mL. These results demonstrate that the concentrations of FA and AA in PET-bottled mineral water on the Japanese market have not changed significantly over the last decade.

A novel fluorescent probe for ratiometric detection of formaldehyde in real food samples, living tissues and zebrafish.

Herein, a two-photon (TP) ratiometric fluorescent probe (NpFA) was developed for detecting formaldehyde (FA) in real food samples, living onion tissues and zebrafish by fluorescence resonance energy transfer (FRET) strategy. Specifically, a TP fluorophore as the donor and a FA turn-on naphthalimide fluorophore as the acceptor were connected by a non-conjugated linker to construct the TP-FRET-based NpFA, which exhibited a target-modulated ratiometric fluorescence response to FA rapidly with high selectivity and sensitivity during 65 s, and a large ratio ~5-fold enhancement at I550/I410 after addition of FA, displaying ~60-fold enhancement at 550 nm and a quite low DOC of 5.8 ± 0.2 nM. Moreover, NpFA has a good imaging resolution and depth of deep tissue penetration. Therefore, based on the above results, NpFA has the capability to be a useful tool for investigating FA in real samples application, and we also hope NpFA will further study of the physiological and pathological function of FA.

One academic year laboratory and student breathing zone formaldehyde level, measured by gas-piston hand pump at gross anatomy laboratory, Siriraj Hospital, Thailand.

This study used a formaldehyde detector tube with a gas-piston hand pump to assess ceiling levels of student breathing zone and gross laboratory environment across the 2018 academic year. The room dimension was 28.6 × 55.48 × 5.5 m. It contained 90 cadavers, each placed on a hinged cover table. We measured before and during nine body region dissections. There was a significant difference (p < 0.01) between student exposure and laboratory environment levels. The highest level was student exposure during body wall dissection (2.7 ppm), the first laboratory; students may accidentally enter body cavities. The latter two were in abdominal (1.85 ppm) and lower limb dissections (1.49 ppm). The three highest environment levels were in different regions; spinal cord removal (1.13 ppm), lower limb (0.72 ppm), and thorax (0.71 ppm) dissection. Only the perineum environment level (0.09 ppm) was below the NIOSH ceiling level (0.1 ppm), which may result from the table covers that had been opened for 2 weeks before measurement. This study finding signified the importance of student personal exposure monitoring and encouraged the academic year measurement. Because each laboratory has unique factors, those affect formaldehyde levels; dissection steps, dissection table design, cadaver storage protocol, and heating-ventilation-air conditioning system performance, for instance.

Highly selective chemosensor for reactive carbonyl species based on simple 1,8-diaminonaphthalene.

Reactive carbonyl species (RCSs) including one carbon formaldehyde (FA) and dicarbonyl compounds such as methylglyoxal (MGO) and glyoxal (GO) are produced during demethylase reactions and various glucose metabolic pathways respectively. Elevation of the RCSs concentrations in cells is due to abnormal DNA damage, glycation adducts with macromolecules that lead to various neurotoxic diseases. Hence, regular monitoring of these RCSs with an easy tool is of utmost interest. However, conventional methods such as chromatography and mass spectrometry for the detection of these species are not so economically viable. These issues were well addressed by the non-invasive reactivity-based fluorescence techniques. However, tedious synthesis, only specific to either mono aldehyde is limited to detect multiple RCSs in physiologies by synthesized fluorophores. An alternative, simple small molecules are widely applied as commercial biomarkers such as terephthalate and 2,3-diaminonaphthalene (NAP) for hydroxy radical (OH·) and nitric oxide (NO) respectively. Herein, we report an analogue of NAP, 1,8-diamino naphthalene (DAN) is an efficient chemosensor for highly sensitive detection of FA, MGO and GO with minimum detection limits of 0.95-3.97 µM. Surprisingly, DAN shows a "turn on" response towards RCSs but remaining silent towards NO which are exactly opposite to commercial probe NAP. Exogenous RCSs imaging in vitro cancerous cells shows the efficacy of the probe and its potential application for RCSs monitoring in cancer cells, generation of toxic byproducts.

New insight into Alzheimer's disease: Light reverses Aß-obstructed interstitial fluid flow and ameliorates memory decline in APP/PS1 mice.

INTRODUCTION: Pharmacological therapies to treat Alzheimer's disease (AD) targeting "Aß" have failed for over 100 years. Low levels of laser light can disassemble Aß. In this study, we investigated the mechanisms that Aß-blocked extracellular space (ECS) induces memory disorders in APP/PS1 transgenic mice and addressed whether red light (RL) at 630 nm rescues cognitive decline by reducing Aß-disturbed flow of interstitial fluid (ISF). METHODS: We compared the heating effects on the brains of rats illuminated with laser light at 630, 680, and 810 nm for 40 minutes, respectively. Then, a light-emitting diode with red light at 630 nm (LED-RL) was selected to illuminate AD mice. The changes in the structure of ECS in the cortex were examined by fluorescent double labeling. The volumes of ECS and flow speed of ISF were quantified by magnetic resonance imaging. Spatial memory behaviors in mice were evaluated by the Morris water maze. Then, the brains were sampled for biochemical analysis. RESULTS: RL at 630 nm had the least heating effects than other wavelengths associated with ~49% penetration ratio into the brains. For the molecular mechanisms, Aß could induce formaldehyde (FA) accumulation by inactivating FA dehydrogenase. Unexpectedly, in turn, FA accelerated Aß deposition in the ECS. However, LED-RL treatment not only directly destroyed Aß assembly in vitro and in vivo but also activated FA dehydrogenase to degrade FA and attenuated FA-facilitated Aß aggregation. Subsequently, LED-RL markedly smashed Aß deposition in the ECS, recovered the flow of ISF, and rescued cognitive functions in AD mice. DISCUSSION: Aß-obstructed ISF flow is the direct reason for the failure of the developed medicine delivery from superficial into the deep brain in the treatment of AD. The phototherapy of LED-RL improves memory by reducing Aß-blocked ECS and suggests that it is a promising noninvasive approach to treat AD.

Exposure to Formaldehyde Perturbs the Mouse Gut Microbiome.

Exposure to Formaldehyde (FA) results in many pathophysiological symptoms, however the underlying mechanisms are not well understood. Given the complicated modulatory role of intestinal microbiota on human health, we hypothesized that interactions between FA and the gut microbiome may account for FA's toxicity. Balb/c mice were allocated randomly to three groups: a control group, a methanol group (0.1 and 0.3 ng/mL MeOH subgroups), and an FA group (1 and 3 ng/mL FA subgroups). Groups of either three or six mice were used for the control or experiment. We applied high-throughput sequencing of 16S ribosomal RNA (rRNA) gene approaches and investigated possible alterations in the composition of mouse gut microbiota induced by FA. Changes in bacterial genera induced by FA exposure were identified. By analyzing KEGG metabolic pathways predicted by PICRUSt software, we also explored the potential metabolic changes, such as alpha-Linolenic acid metabolism and pathways in cancer, associated with FA exposure in mice. To the best of our knowledge, this preliminary study is the first to identify changes in the mouse gut microbiome after FA exposure, and to analyze the relevant potential metabolisms. The limitation of this study: this study is relatively small and needs to be further confirmed through a larger study.

Reduction of Endogenous Melatonin Accelerates Cognitive Decline in Mice in a Simulated Occupational Formaldehyde Exposure Environment.

Individuals afflicted with occupational formaldehyde (FA) exposure often suffer from abnormal behaviors such as aggression, depression, anxiety, sleep disorders, and in particular, cognitive impairments. Coincidentally, clinical patients with melatonin (MT) deficiency also complain of cognitive problems associated with the above mental disorders. Whether and how FA affects endogenous MT metabolism and induces cognitive decline need to be elucidated. To mimic occupational FA exposure environment, 16 healthy adult male mice were exposed to gaseous FA (3 mg/m³) for 7 consecutive days. Results showed that FA exposure impaired spatial memory associated with hippocampal neuronal death. Biochemical analysis revealed that FA exposure elicited an intensive oxidative stress by reducing systemic glutathione levels, in particular, decreasing brain MT concentrations. Inversely, intraperitoneal injection of MT markedly attenuated FA-induced hippocampal neuronal death, restored brain MT levels, and reversed memory decline. At tissue levels, injection of FA into the hippocampus distinctly reduced brain MT concentrations. Furthermore, at cellular and molecular levels, we found that FA directly inactivated MT in vitro and in vivo. These findings suggest that MT supplementation contributes to the rescue of cognitive decline, and may alleviate mental disorders in the occupational FA-exposed human populations.

Validation and Determination of the Contents of Acetaldehyde and Formaldehyde in Foods.

The aim of this study was to develop an efficient quantitative method for the determination of acetaldehyde (AA) and formaldehyde (FA) contents in solid and liquid food matrices. The determination of those compounds was validated and performed using gas chromatography-mass spectrometry combined by solid phase micro-extraction after derivatization with O-(2,3,4,5,6-pentafluoro-benzyl)-hydroxylamine hydrochloride. Validation was carried out in terms of limit of detection, limit of quantitation, linearity, precision, and recovery. Then their contents were analyzed in various food samples including 15 fruits, 22 milk products, 31 alcohol-free beverages, and 13 alcoholic beverages. The highest contents of AA and FA were determined in a white wine (40,607.02 ng/g) and an instant coffee (1,522.46 ng/g), respectively.

Formaldehyde exposure in gross anatomy laboratory of Suranaree University of Technology: a comparison of area and personal sampling.

Cadavers are usually preserved by embalming solution which is composed of formaldehyde (FA), phenol, and glycerol. Therefore, medical students and instructors have a higher risk of exposure to FA inhalation from cadavers during dissection. Therefore, the objective of this study was to evaluate the FA exposure in indoor air and breathing zone of medical students and instructors during dissection classes in order to investigate the relationship between them. The indoor air and personal air samples in breathing zone were collected three times during anatomy dissection classes (in January, August, and October of 2014) with sorbent tubes, which were analyzed by high-performance liquid chromatography (HPLC). The air cleaner machines were determined by weight measurement. Pulmonary function tests and irritation effects were also investigated. The mean of FA concentrations ranged from 0.117 to 0.415 ppm in the indoor air and from 0.126 to 1.176 ppm in the breathing zone of students and instructors. All the personal exposure data obtained exceeded the threshold limit of NIOSH and WHO agencies. The air cleaner machines were not significant difference. The pulmonary function of instructors showed a decrease during attention of classes and statistically significant decreasing in the instructors more than those of the students. Clinical symptoms that were observed in nose and eyes were irritations with general fatigue. We suggested that the modified exhaust ventilation and a locally ventilated dissection work table were considered for reducing FA levels in the gross anatomy dissection room.

Determination of trace amount of formaldehyde base on a bromate-Malachite Green system.

A novel catalytic kinetic spectrophotometric method for determination of trace amount of formaldehyde (FA) has been established, based on catalytic effect of trace amount of FA on the oxidation of Malachite Green (MG) by potassium bromate in presence of sulfuric acid medium, and was reported for the first time. The method was monitored by measuring the decrease in absorbance of MG at 617 nm and allowed a precise determination of FA in the range of 0.003-0.08 µg mL(-1), with a limit of detection down to 1 ng mL(-1). The relative standard deviation of 10 replicate measurements was 1.63%. The method developed was approved to be sensitive, selective and accurate, and adopted to determinate free FA in samples directly with good accuracy and reproducibility.