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Behavior of 1,3-dichloropropene and methyl isothiocyanate in undisturbed soil columns.

To develop alternatives to methyl bromide (MeBr) for soil disinfection under environmental Moroccan conditions, distribution and persistence of 1,3-dichloropropene (1,3-D) and methyl isothiocyanate (MITC) were tested in undisturbed soil columns (12 cm internal diameter, 1 m length). 1,3-D was injected at a 15 cm depth and directly followed by metam-sodium (a precursor of MITC), which was applied at the soil surface of the same column using a peristaltic pump. Concerning the distribution of these fumigants in the soil profiles, our results showed that 24 h after treatment, 1,3-D and MITC were concentrated at the 0-40 cm soil layers, and reached the deeper layers 48 h later. MITC and 1,3-D dissipation was studied and the half-life (DT50) measured were 6.5 and 8 days, respectively. Total volatilization losses reached 9% for MITC and 28% for 1,3-D. MITC and 1,3-D volatilization was found to be influence by soil water contents. The results show that by reducing volatilization, photodegradation and leaching of these fumigants a suitable alternative to MeBr use is offered.

Allyl Compounds↗

The effects of intravenous naltrindole and naltrindole 5'-isothiocyanate on sufentanil-induced respiratory depression and antinociception in rats.

Although the interactions between the mu- and the delta-opiate receptor subtypes are well documented with regard to supraspinal analgesia, less is known about the mutual interactions on respiratory depression. To clarify the functional interactions between both opiate receptor subtypes with regard to antinociception and respiratory depression, male Wistar rats were intravenously injected with 2.5 microg/kg of the mu-opiate agonist sufentanil and subsequently intravenously challenged with the delta antagonist naltrindole (NTI) or naltrindole 5'-isothiocyanate (5'-NTII), a delta-2 antagonist. Antinociception was measured by means of the tail-flick latency, and respiratory depression was evaluated by means of analysis of PaCO2, PaO2, and oxygen saturation. To quantify the antagonistic properties of NTI and 5'-NTII, mean areas under the curve were calculated for groups treated with sufentanil, control vehicle, and sufentanil plus a dose of the antagonists. NTI, but not 5'-NTII, antagonized the sufentanil-induced antinociception at 10 mg/kg NTI. Below this dose the effects were inconsistent. The sufentanil-induced hypercapnia and hypoxia were diminished with 10 mg/kg NTI or 5'-NTII. These data indicate that NTI antagonizes the sufentanil-induced antinociception and respiratory depression in rats. A dissociation between the antinociception and respiratory depression following intravenous sufentanil could be obtained with 10 mg/kg 5'-NTII pointing to different regulatory effects of opiate delta receptor subtypes on mu-opiate agonist-induced behavioral effects.

Analgesics, Opioid↗

Phenethyl isothiocyanate modulates clastogenicity of mitomycin C and cyclophosphamide in vivo.

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is an effective chemopreventive agent against N-nitrosamine-induced carcinogenesis. We have investigated the extent to which PEITC modulates the clastogenicity of standard genotoxicants, mitomycin C and cyclophosphamide, using bone marrow cells of Swiss albino mice. PEITC, 1 mumol/kg body weight in corn oil was administered by gavage for 7 consecutive days to prime the animals. 24 h later, mice received a single dose of cyclophosphamide (10 or 20 mg/kg body weight) or mitomycin C (1 or 2 mg/kg body weight) intraperitoneally. Clastogenicity of the chemicals was compared using PEITC-primed and non-primed animals 24 h after clastogen treatment. As a single agent, PEITC is not clastogenic even after 7 days of priming. Oral priming with PEITC decreased the aberrations per cell values by 22-67% in all cases. PEITC could only alleviate the clastogenicity of 1 mg/kg body weight mitomycin C to near-control values (p < or = 0.05). Although PEITC is reported to be effective against N-nitrosamine-induced tumorigenesis by preventing metabolic activation and by blocking the reactive species formed, it is virtually ineffective against the clastogenicity of cyclophosphamide. The results of inhibition by PEITC of the clastogenicity of mitomycin C suggest that the modulation of mitomycin C bio-activation contributes to, but may not be sufficient for, PEITC chemoprevention of clastogenicity by mitomycin C.

Animals↗

Fluorescein isothiocyanate staining and characterization of avian heterophils.

Fluorescein isothiocyanate (FITC) was found to stain cytoplasmic granules of avian heterophilgranulocytes. In tissue sections, the fluorescent granulocytes were predominantly distributed adjacent to trabecular bones. The fluorescein stained granulocytes were abundant in synovial fluids of chickens with synovitis. A significant correlation was observed in the percent of fluorescein labeled granulocytes in blood smears and the percent of heterophils determined using an automated counting method, in unstained blood from normal and Escherichia coli-infected turkeys. The fluorescein-binding heterophils purified from chickens showed a time dependent increases in the oxidation of 2',7'-dichlorofluorescin diacetate (DCF-DA) and the reduction of nitroblue tetrazolium (NBT) which were indicative of changes in oxidative burst in response to phorbol 12-myristate 13-acetate (PMA), Salmonella typhimurium lipopolysaccharide (LPS), and zymosan A (ZA). These heterophil-activating agents, also, caused significant degranulation at 16 h post-treatment, as indicated by the loss fluorescence. There were microscopically visible alterations in the cell shapes and a decrease in the density of granules due to treatment with LPS, PMA or ZA. In addition, these cells also showed phagocytic response which was evident at 30 min of incubation with fluorescent latex particles. Both chicken and turkey heterophils produced interleukin-6 in vitro at 24 h in response to LPS but not to PMA, FMLP or ZA. The chicken heterophils showed spontaneous production of matrix metalloproteinases (MMP) which was significantly enhanced by treatment with LPS, PMA, and ZA; however, LPS appeared to be most effective in inducing MMP production. These results demonstrate that the functions of heterophils can be differentially regulated by different activating agents and the fluorescein binding property of these cells may be useful for their histochemical identification.

Animals↗

Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds.

Volatile compounds occurring in the essential oil of plants were tested for their efficacy in killing Escherichia coli O157:H7. Experiments using an agar disk assay revealed that exposure of the pathogen to 50 microl of eugenol, carvacrol, linalool, or methyl jasmonate in a 950-cc jar at 20, 37 or 47 degrees C for up to 48 h failed to inhibit colony formation. However, exposure to 8 microl of allyl isothiocyanate (AIT) (equivalent to 8.4 ppm in the air within the jar, if completely volatilized) resulted in more than a 7-log10 reduction in population of E. coli O157:H7 at 37 degrees C within 48 h; significant (P < or = 0.05) reduction in populations also occurred in the presence of 4 microl of AIT compared to 2 microl, which had no lethal affect. At 20 degrees C, the lethality of AIT was substantially less, although significant reduction occurred when disks were exposed to 8 or 10 microl of AIT compared to 4 or 6 microl and when exposed to 4 or 6 microl compared to 2 microl. Treatment with 10 microl of AIT for 5 h at 47 degrees C resulted in death of 6 log10 of E. coli O157:H7. The efficacy of AIT in killing E. coli O157:H7 on dry and wet alfalfa seeds was investigated. The pathogen, at an initial population of 2.7 log10 cfu/g of seed, was not recovered by direct plating (< 0.7 log10 cfu/g) or enrichment of wet seeds exposed to 50 microl of AIT/950-cc jar for 24 h at 37 or 47 degrees C. Exposure of dry seeds containing 2.9 log10 cfu of E. coli O157:H7 per g to an atmosphere containing 100 microl of AIT/950-cc jar (ca. 105 ppm AIT if completely volatilized) for 24 h at 47 degrees C did not eliminate viable E. coli O157:H7 cells. Unfortunately, the enhanced effectiveness of AIT in killing the pathogen on wet alfalfa seeds is offset by a dramatic reduction in seed viability. Nevertheless, the use of AIT as an alternative to chlorine for the purpose of killing E. coli O157:H7 and perhaps other pathogens on alfalfa seed holds promise. Factors that may influence conditions rendering increased sensitivity of E. coli O157:H7 to AIT without compromising seed viability should be investigated.

Escherichia coli O157↗

Evaluation of urinary N-acetyl cysteinyl allyl isothiocyanate as a biomarker for intake and bioactivity of Brussels sprouts.

Isothiocyanates (ITC), glucosinolate hydrolysis products from Brussels sprouts (BS) and other cruciferous vegetables, are considered to protect the body from cancer by induction of detoxification enzymes such as quinone reductase (QR). Urinary N-acetyl-cysteine (NAC) conjugates of ITC have been proposed as biomarkers of crucifer intake. Here we asked if dietary intake and induction of detoxification enzymes are dose-related to urinary NAC conjugate appearance. Male F344 rats (4/group) received an AIN 76B-40 diet containing 0, 10 or 20% freeze-dried BS for 6 days. A human subject ingested 500 g BS. Urinary AITC-NAC was identified in human and rat urine. Ten and 20% BS diets caused a 1.4- and 2.3-fold induction of QR in the pancreas, a 1.5- and 2.5-fold induction in liver and a 3.1- and 3.6-fold induction in colonic epithelium, respectively. Liver and pancreatic QR induction was dose-related, whereas induction of QR in colon was less different between the two doses. Excretion of the conjugate was dose-related only on day 1, and unrelated to dose after day 2. These results suggest that urinary NAC-AITC is a qualitative biomarker for ingestion and bioactivity of BS, but that it may not be dose-related when rats are fed continuously for 2 or more days.

Acetylcysteine↗

The disposition of allyl isothiocyanate in the rat and mouse.

The urine was the major route of excretion of radioactivity (50-80% of dose) following the oral administration (2.5 and 25 mg/kg body weight) of allyl[14C]isothiocyanate (AITC) to male and female Fischer 344 rats and B6C3F1 mice. Smaller amounts were found in the faeces (6-12%) and expired air (3-7%). The major difference between the two species was the greater retention of radioactivity after 4 days within rats (18-24% of dose) when compared with mice (2-5% of dose). Three radioactive components were found in the urine of mice and two in rats. The three components were inorganic thiocyanate, allylthiocarbamoylmercapturic acid and allylthiocarbamoylcysteine in mice, but no cysteine conjugate was found in rat urine. In the mouse, approximately 80% of the 14C was present in the urine as the thiocyanate ion whereas in the rat some 75% was as the mercapturate. This indicates that in the mouse, hydrolysis of AITC was the major metabolic pathway whereas in the rat glutathione conjugation was the major route. A species difference was seen in the amount of [14C]AITC-derived radioactivity present in the whole blood of rats and mice; measurable levels of radioactivity remained within rat blood for a longer time period (up to 240 hr) when compared with mice (96 hr). Examination of the urinary bladders of male and female rats following oral dosing with [14C]AITC showed a sex difference with greater amounts of [14C]AITC and/or its metabolites within the bladder tissue of male rats. This data is discussed in terms of the known species- and sex-specificity of the urinary bladder tumours, which occurred after long-term administration to male rats, but not to female rats or mice of either sex, in a carcinogenicity study conducted by the National Toxicology Program in the USA.

Animals↗

Effect of nicotine, cotinine and phenethyl isothiocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism in the Syrian golden hamster.

The effect of nicotine, cotinine and phenethyl isothiocyanate (PEITC) on metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was studied in the Syrian golden hamster. Urinary metabolite profiles were determined in 24 h urine after a single subcutaneous (s.c.) administration of [5-(3)H]NNK (80 nmol/kg, s.c.). Co-administration of either a 500-fold higher dose of nicotine (40 micromol/kg, s.c.) or a 5000-fold higher dose of cotinine (400 micromol/kg, s.c.) significantly (P<0.001) reduced metabolic activation of NNK by alpha-hydroxylation to 85 and 71% of control, respectively. Co-administration of a 300-fold higher dose of PEITC (1 micromol/g diet) slightly reduced alpha-hydroxylation of NNK (94% of control). Metabolism of NNK by reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased by nicotine (155%), and significantly increased by cotinine (670%, P<0.001) and PEITC (219%, P<0.01). Detoxification of NNAL by glucuronidation was also increased by all three test agents. Detoxification of NNK and NNAL by N-oxidation was marginally increased by nicotine, reduced by PEITC, and significantly reduced by cotinine. The urinary metabolite profiles suggest that nicotine, which occurs in concentrations up to 30000-fold higher than NNK in mainstream cigarette smoke, and cotinine, its proximal metabolite, may have a significant protective effect against in vivo metabolic activation of NNK.

Animals↗

Dissociation equilibria of the tryptophan synthase alpha 2 beta 2 complex in saline buffer and guanidine isothiocyanate, as studied by sedimentation equilibrium.

The dissociation equilibria of Salmonella typhimurium tryptophan synthase alpha 2 beta 2 complex were studied via centrifugation of the complex to sedimentation equilibrium in neutral saline buffers containing 0 to 137 mM guanidine isothiocyanate (GuSCN). The resulting concentration gradients were analyzed in the context of an equilibrium model for sequential dissociation of two alpha subunits from a stable beta 2 subunit. Under the conditions of these experiments, the first dissociation constant alone could be evaluated at GuSCN concentrations < or = 100 mM, and the second dissociation constant alone could be evaluated at GuSCN = 137 mM. At intermediate GuSCN, both dissociation constants were sufficiently well defined to rule out the presence of a large equilibrium cooperative effect in the stepwise dissociation of the alpha subunits.

Bacterial Proteins↗

The effect of 6-methylthiohexyl isothiocyanate isolated from Wasabia japonica (wasabi) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-buatnone-induced lung tumorigenesis in mice.

The present study was undertaken to estimate the effect of 6-methylthiohexyl isothiocyanate (6MHITC) isolated from Wasabia japonica (wasabi) pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in mice. Pretreatment with 6MHITC for 4 consecutive days at a daily dose of 5 micromol significantly inhibited NNK-induced O(6)-methylguanine formation in lungs at 4 h after the injection. In conjugation with this inhibitory effect, 6MHITC suppressed the increase in proliferating nuclear cell antigen level as well as ornithine decarboxylase activity at a promotion stage of NNK-induced lung tumorigenesis. Finally, this treatment of 6MHITC suppressed the NNK-induced lung tumorigenesis in mice. These results suggest that 6MHITC inhibits the development of lung tumors in mice treated with NNK, due to the suppression of initiation stage.

Animals↗

Transcriptional regulation of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of wasabi (Eutrema wasabi Maxim).

Wasabi is a very popular pungent spice in Japan. This study examined the ability of 6-(methylsufinyl)hexyl isothiocyanate (6-MITC), an active principle of wasabi, to induce the cellular expression of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase (QR) in Hepa 1c1c7 cells. The cells were treated with various concentrations of 6-MITC, and were then assessed for cell growth, QR activity and QR mRNA expression. The induction of QR activity and QR mRNA expression was time- and dose-responsive over a narrow range of 0.1-5 microM, with declining induction at higher concentrations due to cell toxicity. Furthermore, transfection studies demonstrated that the induction of transcription of the QR gene by 6-MITC involved an antioxidant/electrophile-responsive element (ARE/EpRE) activation. Our results suggest a novel mechanism by which dietary wasabi 6-MITC may be implicated in cancer chemoprevention.

Animals↗

Inhibitory effects of 2-mercaptoethane sulfonate and 6-phenylhexyl isothiocyanate on urinary bladder tumorigenesis in rats induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

The effects of 2-mercaptoethane sulfonate (MESNA) and phenethyl and 6-phenylhexyl isothiocyanates (PEITC and PHITC) on urinary bladder tumorigenesis were investigated in Wistar rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN). A total of 190 male rats were divided into eight groups. Animals in groups 1, 3 and 4 were administered BHBN in drinking water (0.025%) and group 2 rats were given 0.025% BHBN plus 0.05% MESNA solution for 15 weeks. Rats in groups 3 and 4 were fed a diet supplemented with PEITC or PHITC at a dose level of 0.5 micromol/g for 16 weeks starting 1 week prior to the BHBN treatment. Rats in groups 5-7 served as MESNA, PEITC or PHITC alone controls. Group 8 served as a non-treatment control. The chromatographic profile of drinking water for group 2 animals indicated no chemical interaction between BHBN and MESNA. The incidences of papillomas in the bladder and total bladder tumors including transitional cell carcinomas were significantly (P<0.01) reduced in group 2 as compared to that in group 1. The multiplicities of papillomas or total bladder tumors were significantly (P<0.01) lower in group 2 and 4 rats treated with MESNA and PHITC, respectively, than in group 1 rats. The PEITC treatment, however, did not affect the BHBN-induced bladder tumorigenesis. No bladder tumors were found in animals of groups 5-8. The results in the present study clearly indicate that both MESNA and PHITC are potent chemopreventive agents against bladder tumor development in rats induced by BHBN.

Animals↗

Benzyl isothiocyanate: an effective inhibitor of polycyclic aromatic hydrocarbon tumorigenesis in A/J mouse lung.

Polycyclic aromatic hydrocarbons (PAH) are an important group of carcinogens that are likely to be involved as one of the causes of lung cancer in smokers and occupationally exposed individuals. Previous studies have shown that benzyl isothiocyanate (BITC), administered by gavage, is a good inhibitor of lung tumorigenesis in A/J mice induced by benzo[a]pyrene (BaP), a typical PAH carcinogen. In this study, we evaluated the effects of BITC on lung tumor induction in A/J mice by two other carcinogenic PAH in cigarette smoke - 5-methylchrysene (5-MeC) and dibenz[a,h]anthracene (DBahA). We also compared the effects of BITC with two other well known chemopreventive agents - butylated hydroxyanisole (BHA) and sulforaphane. In experiment 1, groups of A/J mice were treated by gavage once weekly for 8 weeks with BaP (3 micromol) or 5-MeC (2 micromol) or DBahA (1 micromol) in 0.1 ml cottonseed oil. Fifteen minutes before each treatment, the mice were gavaged with 0.1 ml cottonseed oil or 0.1 ml cottonseed oil containing 13.4 micromol or 6.7 micromol of BITC. The experiment was terminated 19 weeks after the final carcinogen treatment. BITC significantly reduced lung tumor multiplicity in all PAH-treated groups by 63.5-90.6%. In experiment 2, groups of A/J mice were treated with BaP or BITC and BaP as in experiment 1, or with BHA or sulforaphane at doses equimolar to those of BITC. BITC was significantly more effective as an inhibitor of lung tumor induction than either BHA or sulforaphane. These results firmly establish gavaged BITC as a strong inhibitor of lung tumorigenesis induced in A/J mice by PAH, and support its further development for chemoprevention of smoking-induced lung cancer.

Animals↗

Failure of phenethyl isothiocyanate to inhibit hamster tumorigenesis induced by N-nitrosobis(2-oxopropyl)amine when given during the post-initiation phase.

The chemopreventive influence of phenethyl isothiocyanate (PEITC) during the post-initiation stage was investigated in the N-nitrosobis(2-oxopropyl)amine (BOP)-initiated hamster tumorigenesis model. A total of 120 female 5-week-old hamsters were divided into six groups. Animals in groups 1-3, each consisting of 30 hamsters, were injected twice, subcutaneously, with BOP 7 days apart to effect initiation. Starting 1 week after the second BOP injection, hamsters in groups 1 and 2 were fed diets supplemented with 6 micromol/g and 3 micromol/g of PEITC, respectively, for 51 weeks. Animals in group 3 received a basal diet as an initiation positive control. Animals in groups 4-6, each consisting of ten hamsters, were given 6 micromol/g or 3 micromol/g of PEITC alone, or were non-treated, matched negative controls for groups 1-3. At the termination of experimental week 52, the incidences and multiplicities of neoplastic lesions in the target organs including the pancreas, lung, liver and kidney were found to be comparable among the BOP-treated groups. The values for pancreatic adenocarcinomas as well as dysplastic lesions tended to increase although without statistical significance. Taken together with our previous finding that PEITC dramatically inhibited the initiation phase of BOP-induced pancreatic and lung tumorigenesis in hamsters, it can be concluded that PEITC specifically exerts chemopreventive effects only when given concomitantly with the carcinogen.

Adenocarcinoma↗

In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi.

Sulfur K-edge X-ray absorption spectroscopy has been used to determine the chemical identity of the sulfur-containing species in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) in situ, before and after cell disruption. The major sulfur-containing species in the intact root is sinigrin (1-thio-beta-D-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners. Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in sinigrin to isothiocyanates and sulfate in approximately equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed, but an unusual thio intermediate may have been identified for the first time.

Brassica↗

Barbarea verna as a source of 2-phenylethyl glucosinolate, precursor of cancer chemopreventive phenylethyl isothiocyanate.

The isolation of gram-amounts of 2-phenylethyl glucosinolate (gluconasturtiin, GST) from Barbarea verna seeds is reported for the first time. This vegetable source was of crucial importance to isolate GST with a high purity grade and in high yield. Indeed, B. verna seeds contain GST as the only glucosinolate, unlike other sources. The availability at low cost of GST will allow further studies to explain the claimed anticancer activity of its derived phenylethyl isothiocyanate.

Aldehyde Dehydrogenase↗

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatizaton with 2,3,4,6,-tetra-O-acetyl-beta-D-glycopyranosyl isothiocyanate.

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-beta-D-glycopyranosyl isothiocyanate at 35 degrees C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)-acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 microm particle size, 10 cm x 0.46 cm I.D.) with a mobile phase of phosphate buffer-methanol-acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV-VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 microg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 microg/ml (RSD <10%). In the range 0.145-20 microg/ml, intra-day coefficients of variation were 1.0-7.0% and inter-day coefficients of variation were 0.4-16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.

Adrenergic beta-Antagonists↗

Determination of tranexamic acid in human serum by high-performance liquid chromatography using selective pre-column derivatization with phenyl isothiocyanate.

A high-performance liquid chromatographic method for determination of tranexamic acid in human serum using a selective derivatization has been developed. Tranexamic acid in the sample was allowed to react with phenyl isothiocyanate to form the phenylthiocarbamoyl derivative. Interfering alpha-amino acids in the sample were eliminated by selective derivatization to phenylthiohydantoin derivatives by acid treatment of the phenylthiocarbamoyl derivatives followed by solvent extraction. Then, the sample was analysed by conventional high-performance liquid chromatography with ultraviolet detection. The limit of detection of this method for serum sample was 0.2 micrograms/ml at a signal-to-noise ratio of 2. This method gave results comparable with those obtained by amino acid analysis (regression line: y = 0.4531x-0.02596, r = 0.9998, n = 21).

Amino Acids↗