Biomedical subjects

A D Jones

Publications and source records attributed to A D Jones.

At least 73 records · Page 4Linked to original sources

PubMed1996

Simultaneous determination of multiple aldehydes in biological tissues and fluids using gas chromatography/stable isotope dilution mass spectrometry.

Biological oxidative stress has been associated with various degenerative disorders and disease states, and accurate and sensitive methods are needed to determine the extent of oxidation occurring in vivo. Peroxidation of polyunsaturated fatty acids forms complex mixtures of aldehydes and other breakdown products because various oxidants are involved and lipid composition is not uniform. Quantitative analysis of multiple lipid peroxidation products yields a more complete measure of biological oxidation than measurement of a single aldehyde, particularly when aldehydes exhibit marked differences in reactivity. This report describes extensions of an established gas chromatography/mass spectrometry method to include stable isotope dilution determination in tissue and plasma of three aldehydic products of lipid peroxidation: hexanal, nonanal, and 4-hydroxy-2-nonenal. Use of deuterated internal standards for each analyte improved precision and accuracy compared to a single internal standard for all three aldehydes. Improvements are attributed to differences in extraction and derivatization efficiencies for individual analytes owing to substantial differences in reactivity. Electron ionization of oxime-tert-butyldimethylsilyl derivatives gave greater specificity for detecting all three aldehydes than was possible using electron-capture ionization of O-pentafluorobenzyl oxime derivatives. Exchange of the deuterium label of [2,3-2H]HNE internal standard was determined to be minimal during the analyses.

Aldehydes↗

PubMed1996

Haloenol lactone is a new isozyme-selective and active site-directed inactivator of glutathione S-transferase.

A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 microM) with murine Alpha, Mu, or Pi GST isozyme (1.0 microM) at pH 6.5, 37 degrees C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, approximately 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37 degrees C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 microM) was incubated with GST Pi isozyme (1.0 microM) in the presence of the competitive inhibitor S-hexylglutathione (10 microM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.

4-Butyrolactone↗

PubMed1996

Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid. A novel mechanism for nitric oxide-mediated protein modification.

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO-). We show here that reaction of nitrite (NO2-), the autoxidation product of nitric oxide (.NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO2- with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO2-/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO2-/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO2-/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO2-/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (.NO2) and tyrosyl radical. We propose that NO2- reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl. and .NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO-.

Hypochlorous Acid↗

PubMed1996

Laser desorption/ionization time-of-flight mass spectrometry of nitrated polycyclic aromatic hydrocarbons.

Mass spectra of four nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), 9-nitroanthracene, 1-nitropyrene, 2-nitro-9-fluorenone, and 2-nitrofluorene, have been investigated using single-step laser desorption/ionization time-of-flight mass spectrometry. Pulsed UV radiation at 266 or 213 nm was used for desorption and ionization of solid samples deposited on an aluminum probe. The positive molecular ion which was observed for each nitro-PAH was of greater relative intensity when 213 nm radiation was used. A strong [M - NO]+ peak was observed in all spectra, and an intense NO+ signal accompanied the [M - NO]+ signal when 213 nm was used but was only weakly present when 266 nm was used. Comparison of the various spectra suggests that nitro-PAHs undergo an excited state nitro-nitrite rearrangement, followed by loss of NO. Multiphoton ionization of the NO fragment appears to be the principal route of formation of NO+ during laser desorption/ionization when 213 nm radiation is used. The presence of the carbonyl group in 2-nitro-9-fluorenone leads to unique and prominent fragments involving losses of CO from the carbonyl bridge.

Air Pollutants↗

PubMed1996

Evidence of overload, dissolution and breakage of MMVF10 fibres in the RCC chronic inhalation study.

This paper describes (i) the evidence revealing the biological mechanisms underlying data from inhalation experiments at the Research and Consulting Company (RCC) (HESTERBERG et al. 1993); and (ii) the quantitative evaluation of the kinetic rates of these mechanisms using a mathematical model developed at the Institute of Occupational Medicine (IOM) (TRAN et al. 1994).

Administration, Inhalation↗

PubMed1996

Rapid assays for environmental and biological monitoring.

Rapid, inexpensive, sensitive, and selective enzyme-linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s-triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury.

Antifungal Agents↗

PubMed1996

Dioxinlike properties of a trichloroethylene combustion-generated aerosol.

Conventional chemical analyses of incineration by-products identify compounds of known toxicity but often fail to indicate the presence of other chemicals that may pose health risks. In a previous report, extracts from soot aerosols formed during incomplete combustion of trichloroethylene (TCE) and pyrolysis of plastics exhibited a dioxinlike response when subjected to a keratinocyte assay. To verify this dioxinlike effect, the complete extract, its polar and nonpolar fractions, some containing primarily halogenated aromatic hydrocarbons, were evaluated for toxicity using an embryo assay, for antiestrogenicity using primary liver cell cultures, and for the ability to transform the aryl hydrocarbon receptor into its DNA binding form using liver cytosol in a gel retardation assay. Each of these assays detect dioxinlike effects. Medaka (Oryzias latipes) embryos and primary liver cell cultures of rainbow trout (Oncorhynchus mykiss) were exposed to concentrations of extract ranging from 0.05 to 45 micrograms/l. Cardiotoxicity with pericardial, yolk sac, and adjacent peritoneal edema occurred after exposure of embryos to concentrations of 7 micrograms/l or greater. These same exposure levels were associated with abnormal embryo development and, at the higher concentrations, death. Some of the fractions were toxic but none was as toxic as the whole extract. In liver cells, total cellular protein and cellular lactate dehydrogenase activity were not altered by in vitro exposure to whole extract (0.05-25 micrograms/l). However, induction of cytochrome P4501A1 protein and ethoxyresorufin O-deethylase activity occurred. In the presence of whole extract, estradiol-dependent vitellogenin synthesis was reduced. Of the fractions, only fraction 1 (nonpolar) showed a similar trend, although vitellogenin synthesis inhibition was not significant. The soot extract and fractions bound to the Ah receptor and showed a significantly positive result in the gel retardation/DNA binding test. Chemical analyses using GC-MS with detection limits for 2,3,7,8-tetrachlorodibenzo-p-dioxin and dibenzofuran in the picomole range did not show presence of these compounds. Our results indicate that other chemicals associated with TCE combustion and not originally targeted for analysis may also pose health risks through dioxinlike mechanisms.

Aerosols↗

PubMed1995

Mechanism of soluble epoxide hydrolase. Formation of an alpha-hydroxy ester-enzyme intermediate through Asp-333.

18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H2 18O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 180. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an alpha-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.

Animals↗

PubMed1995

Molecular and biochemical evidence for the involvement of the Asp-333-His-523 pair in the catalytic mechanism of soluble epoxide hydrolase.

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

Amino Acid Sequence↗

PubMed1995

Formation of epoxide and quinone protein adducts in B6C3F1 mice treated with naphthalene, sulfate conjugate of 1,4-dihydroxynaphthalene and 1,4-naphthoquinone.

Naphthalene (NA) is metabolically activated to the reactive intermediates, naphthalene oxide (NO) and naphthoquinones. To investigate the role of circulating reactive metabolites in NA toxicity, the half-life of NO was examined. The in vitro half-life of NO in both whole blood and plasma was 10 min. Detectable levels of NO were seen in perfusate leaving the isolated perfused liver of B6C3F1 mice infused with 10 mumol/h NA. Identification of protein sulfhydryl adducts in NA-exposed mice (50 and 100 mg/kg, IP, 24 h) revealed a predominance of quinone adducts in liver, lung, kidney, red blood cells and brain. The epoxide adduct predominated in plasma protein. Administration of the sulfate conjugate of 1,4-dihydroxynaphthalene (NHQS) (100 mg/kg) resulted in formation of naphthoquinone protein sulfhydryl adducts in lung, liver and kidney. Administration of 1,4-naphthoquinone (NQ) (5 mg/kg) produced NQ adducts in liver, lung, kidney, plasma and brain.

Animals↗

PubMed1995

Direct characterization of protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal using electrospray mass spectrometry.

Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.

Aldehydes↗

PubMed1995

Purification, mass spectrometric characterization, and covalent modification of murine glutathione S-transferases.

Three cytosolic glutathione S-transferase (GST, EC 2.5.1.18) isozymes were purified from livers of male CD-1 mice to homogeneity using affinity chromatography and gradient elution. Isozyme molecular masses and purities were determined using electrospray ionization mass spectrometry (ESI/MS), HPLC, and electrophoretic methods. Isozymes were assigned to alpha, pi, and mu classes based on kinetic and electrophoretic properties. Molecular masses determined by mass spectrometry were 25 271.4 +/- 2, 23 478.8 +/- 2, and 25 838.4 +/- 2 Da for alpha, pi, and mu isozymes, respectively. Molecular masses for pi and mu class isozymes agreed with molecular masses derived from nucleic acid sequences, but the molecular mass of the alpha class isozyme was 42 Da greater than calculated for a reported sequence. Analysis of tryptic digests of GST-alpha using tandem mass spectrometry determined that the molecular weight discrepancy could be attributed to N-terminal acetylation. Effects of covalent attachment of 4-hydroxy-2-nonenal (HNE) on catalytic activities of each GST isozyme were investigated by incubating GSTs with HNE. The extent of HNE-protein adduct formation was determined using ESI/MS. Catalytic activity of HNE-modified GSTs toward CDNB was measured. ESI mass spectra of modified enzymes show that the extent of HNE modification to the isozymes (pi > alpha > mu) coincides with the loss of activity seen for each protein. ESI mass spectra also indicated that glutathione-protein adducts form in isozymes during incubations with HNE even though purified proteins had been dialyzed against water before incubations. Glutathionylation of isozymes increased as the concentration of HNE in the incubations was raised. Dialysis of GSTs against 2 mM S-hexylglutathione (to displace GSH) before incubations eliminated S-glutathionylation. Reactivities of these GSTs toward HNE differed and is related to the number of cysteine residues (alpha < mu < pi) within the amino acid sequence.

Animals↗

PubMed1995

The sizes, shapes, and mineralogy of asbestos structures that induce lung tumors or mesothelioma in AF/HAN rats following inhalation.

Data from inhalation studies in which AF/HAN rats were exposed to nine different types of asbestos dusts (in 13 separate experiments) are employed in a statistical analysis to determine if a measure of asbestos exposure (expressed as concentrations of structures with defined sizes, shapes and mineralogy) can be identified that satisfactorily predicts the observed lung tumor or mesothelioma incidence in the experiments. Due to limitations in the characterization of asbestos structures in the original studies, new exposure measures were developed from samples of the original dusts that were re-generated and analyzed by transmission electron microscopy using a direct transfer technique. This analysis provided detailed information on the mineralogy (i.e., chrysotile, amosite, crocidolite or tremolite), type (i.e., fiber, bundle, cluster, or matrix), size (length and width) and complexity (i.e., number of identifiable components of a cluster or matrix) of each individual structure. No univariate measure of exposure was found to provide an adequate description of the lung tumor responses observed among the inhalation studies, although the measure most highly correlated with tumor incidence is the concentration of structures > or = 20 microns in length. Multivariate measures of exposure were identified that do adequately describe the lung tumor responses. Structures contributing to lung tumor risk appear to be long (> or = 5 microns) thin (0.4 microns) fibers and bundles, with a possible contribution by long and very thick (> or = 5 microns) complex clusters and matrices. Potency appears to increase with increasing length, with structures longer than 40 microns being about 500 times more potent than structures between 5 and 40 microns in length. Structures < 5 microns in length do not appear to make any contribution to lung tumor risk. This analysis did not find a difference in the potency of chrysotile and amphibole toward the induction of lung tumors. However, mineralogy appears to be important in the induction of mesothelioma with chrysotile being less potent than amphibole.

Administration, Inhalation↗

PubMed1995

Protocol for collection and HPLC analysis of volatile carbonyl compounds in breath.

This noninvasive method for collection and analysis of a wide range of aldehydes and ketones in human breath may enable assessment of lipid peroxidation and metabolic status in vivo. Breath samples are drawn through silica cartridges impregnated with 2,4-dinitrophenylhydrazine, which traps carbonyls as their hydrazone derivatives. The hydrazone derivatives are eluted from the cartridges with acetonitrile, separated by reversed-phase HPLC, and quantified spectrophotometrically. Using this method, we have measured formaldehyde, acetaldehyde, acetone, propanal, 2-butanone, butanal, pentanal, and hexanal. Recoveries of carbonyls added to Douglas bags were > 90%, except for 2-butanone, which was 86.2%. The overall CVs for sampling plus analyzing duplicate aliquots of breath were < 11%. The results indicate that this protocol can be used to monitor changes of carbonyl production by analyzing expired air, which may, with further study, indicate physiological and pathological status.

Acetaldehyde↗

PubMed1995

Biliary excretion of pyrrolic metabolites of [14C]monocrotaline in the rat.

The biliary excretion of amphoteric pyrroles was studied using in situ isolated rat liver preparations perfused in a recirculatory manner for 60 to 90 min with 14C-monocrotaline (40 mumol/120 ml of Krebs-Henseleit buffer). After 90 min, 20% of the administered 14C and an amount of pyrrole equivalent to 9 mumol of dehydroretronecine was recovered in the bile. Bile collected between 15 and 30 min contained the highest levels of 14C and pyrrolic metabolites. Isoelectric focusing (IEF) and electrophoretic separations on matrices of polyacrylamide and silica were used to isolate and estimate levels of pyrrolic conjugates. IEF and electrophoretic separations on silica revealed the presence of six pyrrole-positive metabolites. Reactions of monocrotaline pyrrole with glutathione produced three conjugates that had the same pI values as components found in bile. Electrophoretic separations on silica and polyacrylamide followed by reversed phase chromatography removed sufficient biliary matrix contaminants to permit identification of glutathione and cysteinyl-glycine conjugates of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine and 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine with fast atom bombardment MS/MS. This study revealed a complexity in the biliary pyrrolic excretion profile that had not previously been realized.

Animals↗

PubMed1995

Metabolism of [14C]naphthalene in the B6C3F1 murine isolated perfused liver.

Naphthalene (NA) is metabolized by pulmonary and hepatic tissues to epoxides, quinones, and their related phase II metabolites. To delineate specific liver metabolites, metabolism was studied in B6C3F1 mouse liver perfused with 5 and 10 mumol/hr [14C]NA. Liver metabolites were compared with urinary metabolites from mice exposed to an equivalent total dose of NA (50 mg/kg ip) to ascertain interorgan and extrahepatic transformation in vivo. Metabolites were separated into pools via hydrophobic columns under neutral (pool I) and acid (pool II) conditions. Pool I contained the majority of [14C] in perfusate and urine. In perfusate, high levels of sulfate conjugates of naphthol and dihydroxynaphthalene were found along with dihydrodiol and glucuronic acid conjugates. In the urine, dihydrodiol was the most abundant metabolite. A novel N-acetylated glutathione conjugate was a constituent of pool II of both perfusate and urine. Additional metabolites identified in urine were N-acetylcysteine conjugate of dihydrodiol epoxide, mercaptolactic acid conjugate of naphthalene oxide (NO), and diglucuronide and sulfate/glucuronide conjugates of dihydroxynaphthalene. Mercapturic acid conjugates of NO were not observed in either perfusate or urine; this finding highlights metabolic differences between strains. Differential covalent binding occurred in cellular fractions, with the highest binding occurring in microsomes and mitochondria. These metabolites indicate that interorgan metabolism plays a role in the disposition of NA in vivo.

Animals↗

PubMed1995

Mathematical model of phagocytosis and inflammation after the inhalation of quartz at different concentrations.

A mathematical model was developed with several distinctive features to represent the various processes leading to silicosis. It assures the phagocytosis of quartz by the resident alveolar macrophages. It then models the necrosis of macrophages caused by the ingestion of quartz particles and the subsequent release of these clusters onto alveolar surfaces. The inflammatory recruitment of alveolar macrophages and neutrophils in response to the cell necrosis caused by the ingestion of quartz particles is described in a functional form and is included in the model. The model also describes the pathological and other biological reactions to quartz particles in the interstitium. Specifically, once the quartz particles arrive at the interstitium, they can be phagocytozed by interstitial macrophages. The pathological interaction between quartz and interstitial macrophages may lead to quartz particles being sequestered in silicotic nodules, and also cleared to the efferent lymph nodes.

Administration, Inhalation↗

PubMed1994

High alpha-tomatine content in ripe fruit of Andean Lycopersicon esculentum var. cerasiforme: developmental and genetic aspects.

A variant of Lycopersion esculentum var. cerasiforme is described that deviates from the typical form of the entire species, including cultivated tomatoes, in possessing high levels (500-5000 micrograms/g of dry weight) of the steroidal alkaloid alpha-tomatine in its ripe fruits. This biotype is restricted to a tiny enclave in the valley of Río Mayo, Department San Martín, Peru. Among 88 accessions of var. cerasiforme from its present distribution in the Andes, a 90% association was found between high tomatine and bitter flavor; within the Mayo watershed, all samples from the upper drainage had bitterness and high tomatine; the frequency of both traits decreased to low levels toward the lower end. Tomatine therefore probably is the source of bitterness. Throughout L. esculentum tomatine is present at very high concentrations in earliest stages of fruit development, thereafter decreasing rapidly to midperiod, and finally diminishing gradually to near zero at maturity as a result of catabolism to biologically inert compounds, except in the variant described here. High tomatine content does not appear to affect adversely either the natives, among whom the bitter types are popular, or individuals who sampled them in this survey. Genetic determination of high tomatine in ripe fruits is totally recessive and appears to be monogenic with interaction with genes of minor effect. The prevailing pattern of glycoalkaloid synthesis and degradation in development of solanaceous fruits suggests a mechanism to protect against predation prior to ripening but to permit it afterward as a device to promote dispersal. In consideration of the nondegradative nature of the variant, its genetic determination, and very restricted geographic distribution, mutation to this form appears to be a random event of doubtful evolutionary significance.

Chromatography, Gas↗