One-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of formate production. Safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade in MC38 tumors, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

A Transfer Hydrogenation Approach to Activity-Based Sensing of Formate in Living Cells.

Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.

Quantitative profiling of carboxylic compounds by gas chromatography-mass spectrometry for revealing biomarkers of diabetic kidney disease.

Diabetic kidney disease (DKD), a common microvascular complication of diabetes, currently lacks specific diagnostic indicators and therapeutic targets, resulting in miss of early intervention. To profile metabolic conditions in complex and precious biological samples and screen potential biomarkers for DKD diagnosis and prognosis, a rapid, convenient and reliable quantification method for carboxyl compounds by gas chromatography-mass spectrometry (GC-MS) was established with isobutyl chloroformate derivatization. The derivatives were extracted with hexane, injected into GC-MS and quantified with selected ion monitoring mode. This method showed excellent linearity(R2 > 0.99), good recoveries (81.1%-115.5%), good repeatability (RSD < 20%) and sensitivity (LODs: 0.20-499.90 pg, LOQs: 2.00-1007.00 pg). Among the 37 carboxyl compounds analyzed, 12 metabolites in short-chain fatty acids (SCFAs) metabolism pathway and amino acid metabolism pathway were linked with DKD development and among them, 6 metabolites were associated with both development and prognosis of DKD in mice. In conclusion, a reliable, convenient and sensitive method based on isobutyl chloroformate derivatization and GC-MS analysis is established and successfully applied to quantify 37 carboxyl compounds in biological samples of mice and 12 potential biomarkers for DKD development and prognosis are screened.

New Insight into CO2 Reduction to Formate by In Situ Hydrogen Produced from Hydrothermal Reactions with Iron.

To reveal the nature of CO2 reduction to formate with high efficiency by in situ hydrogen produced from hydrothermal reactions with iron, DFT calculations were used. A reaction pathway was proposed in which the formate was produced through the key intermediate species, namely iron hydride, produced in situ in the process of hydrogen gas production. In the in situ hydrogenation of CO2, the charge of H in the iron hydride was -0.135, and the Fe-H bond distance was approximately 1.537 Å. A C-H bond was formed as a transition state during the attack of Hδ- on Cδ+. Finally, a HCOO species was formed. The distance of the C-H bond was 1.107 Å. The calculated free energy barrier was 16.43 kcal/mol. This study may provide new insight into CO2 reduction to formate in hydrothermal reactions with metal.

Enantioselective Synthesis of Chiral Carboxylic Acids from Alkynes and Formic Acid by Nickel-Catalyzed Cascade Reactions: Facile Synthesis of Profens.

We report a stereoselective conversion of terminal alkynes to α-chiral carboxylic acids using a nickel-catalyzed domino hydrocarboxylation-transfer hydrogenation reaction. A simple nickel/BenzP* catalyst displayed high activity in both steps of regioselective hydrocarboxylation of alkynes and subsequent asymmetric transfer hydrogenation. The reaction was successfully applied in enantioselective preparation of three nonsteroidal anti-inflammatory profens (>90 % ees) and the chiral fragment of AZD2716.

Ammonium Formate-Pd/C as a New Reducing System for 1,2,4-Oxadiazoles. Synthesis of Guanidine Derivatives and Reductive Rearrangement to Quinazolin-4-Ones with Potential Anti-Diabetic Activity.

1,2,4-Oxadiazole is a heterocycle with wide reactivity and many useful applications. The reactive O-N bond is usually reduced using molecular hydrogen to obtain amidine derivatives. NH4CO2H-Pd/C is here demonstrated as a new system for the O-N reduction, allowing us to obtain differently substituted acylamidine, acylguanidine and diacylguanidine derivatives. The proposed system is also effective for the achievement of a reductive rearrangement of 5-(2'-aminophenyl)-1,2,4-oxadiazoles into 1-alkylquinazolin-4(1H)-ones. The alkaloid glycosine was also obtained with this method. The obtained compounds were preliminarily tested for their biological activity in terms of their cytotoxicity, induced oxidative stress, α-glucosidase and DPP4 inhibition, showing potential application as anti-diabetics.

Determination of proteinaceous free amino acids by gas chromatography.

A method was developed for determination of proteinaceous free amino acids by gas chromatography-mass spectrometry (GC-MS). The guanidino group of arginine in amino acid samples was modified with 1,2-cyclohexanedione at room temperature under basic conditions, and all amino acids were directly derivatized with isobutyl chloroformate. The amino acid derivatives formed were analyzed by GC-MS. The method developed was applied successfully for the determination of amino acids in the Japanese alcoholic beverage, sake.

Low temperature plasma irradiation products of sodium lactate solution that induce cell death on U251SP glioblastoma cells were identified.

Low-temperature plasma is being widely used in the various fields of life science, such as medicine and agriculture. Plasma-activated solutions have been proposed as potential cancer therapeutic reagents. We previously reported that plasma-activated Ringer's lactate solution exhibited selective cancer-killing effects, and that the plasma-treated L-sodium lactate in the solution was an anti-tumor factor; however, the components that are generated through the interactions between plasma and L-sodium lactate and the components responsible for the selective killing of cancer cells remain unidentified. In this study, we quantified several major chemical products, such as pyruvate, formate, and acetate, in plasma-activated L-sodium lactate solution by nuclear magnetic resonance analysis. We further identified novel chemical products, such as glyoxylate and 2,3-dimethyltartrate, in the solution by direct infusion-electrospray ionization with tandem mass spectrometry analysis. We found that 2,3-dimethyltartrate exhibited cytotoxic effects in glioblastoma cells, but not in normal astrocytes. These findings shed light on the identities of the components that are responsible for the selective cytotoxic effect of plasma-activated solutions on cancer cells, and provide useful data for the potential development of cancer treatments using plasma-activated L-sodium lactate solution.

Characterization of pepcan-23 as pro-peptide of RVD-hemopressin (pepcan-12) and stability of hemopressins in mice.

Hemopressins ((x)-PVNFKLLSH) or peptide endocannabinoids (pepcans) can bind to cannabinoid receptors. RVD-hemopressin (pepcan-12) was shown to act as endogenous allosteric modulator of cannabinoid receptors, with opposite effects on CB1 and CB2, respectively. Moreover, the N-terminally elongated pepcan-23 was detected in different tissues and was postulated to be the pro-peptide of RVD-hemopressin. Currently, data about the pharmacokinetics, tissue distribution and stability of hemopressin-type peptides are lacking. Here we investigated the secondary structure and physiological role of pepcan-23 as precursor of RVD-hemopressin. We assessed the metabolic stability of these peptides, including hemopressin. Using LC-ESI-MS/MS, pepcan-23 was measured in mouse tissues and human whole blood (~50 pmol/mL) and in plasma was the most stable endogenous peptide containing the hemopressin sequence. Using peptide spiked human whole blood, mouse adrenal gland and liver homogenates demonstrate that pepcan-23 acts as endogenous pro-peptide of RVD-hemopressin. Furthermore, administered pepcan-23 converted to RVD-hemopressin in mice. In circular dichroism spectroscopy, pepcan-23 showed a helix-unordered-helix structure and efficiently formed complexes with divalent metal ions, in particular Cu(II) and Ni(II). Hemopressin and RVD-hemopressin were not bioavailable to the brain and showed poor stability in plasma, in agreement with their overall poor biodistribution. Acute hemopressin administration (100 mg/kg) did not modulate endogenous RVD-hemopressin/pepcan-23 levels or influence the endocannabinoid lipidome but increased 1-stearoyl-2-arachidonoyl-sn-glycerol. Overall, we show that pepcan-23 is a biological pro-peptide of RVD-hemopressin and divalent metal ions may regulate this process. Given the lack of metabolic stability of hemopressins, administration of pepcan-23 as pro-peptide may be suitable in pharmacological experiments as it is converted to RVD-hemopressin in vivo.

Gellan gum gel tissue phantoms and gel dosimeters with tunable electrical, mechanical and dosimetric properties.

Gellan gum gels have been proposed as tissue- and water-mimicking materials (phantoms) applied in medical imaging and radiotherapy dosimetry. Phantoms often require ionic additives to induce desirable electrical conductivity, resistance to biological spoilage, and radical scavenging properties. However, gellan gum is strongly crosslinked by the typically used sodium salts, forming difficult-to-work with gels with reduced optical clarity. Herein we investigated lithium and tetramethylammonium chloride to induce the required electrical conductivity while maintaining optical clarity; lithium formate and methylparaben were used as a radical scavenger and antimicrobial additive, respectively. Using a multifactorial design of experiments, we studied and modeled the electrical and mechanical properties and liquid expulsion (syneresis) properties of the gels. Finally, by the addition of a radiation-sensitive tetrazolium salt, dosimeters with favorable properties were produced. The results described herein may be used to prepare tissue phantoms and dosimeters with tuned electrical, mechanical, and dosimetric properties.

Ultrasonic-assisted catalytic transfer hydrogenation for upgrading pyrolysis-oil.

Recent interest in biomass-based fuel blendstocks and chemical compounds has stimulated research efforts on conversion and upgrading pathways, which are considered as critical commercialization drivers. Existing pre-/post-conversion pathways are energy intense (e.g., pyrolysis and hydrogenation) and economically unsustainable, thus, more efficient process solutions can result in supporting the renewable fuels and green chemicals industry. This study proposes a process, including biomass conversion and bio-oil upgrading, using mixed fast and slow pyrolysis conversion pathway, as well as sono-catalytic transfer hydrogenation (SCTH) treatment process. The proposed SCTH treatment employs ammonium formate as a hydrogen transfer additive and palladium supported on carbon as the catalyst. Utilizing SCTH, bio-oil molecular bonds were broken and restructured via the phenomena of cavitation, rarefaction, and hydrogenation, with the resulting product composition, investigated using ultimate analysis and spectroscopy. Additionally, an in-line characterization approach is proposed, using near-infrared spectroscopy, calibrated by multivariate analysis and modeling. The results indicate the potentiality of ultrasonic cavitation, catalytic transfer hydrogenation, and SCTH for incorporating hydrogen into the organic phase of bio-oil. It is concluded that the integration of pyrolysis with SCTH can improve bio-oil for enabling the production of fuel blendstocks and chemical compounds from lignocellulosic biomass.

The solubility of N-acetyl amino acid amides in organic acid and alcohol solutions: Mechanistic insight into structural protein solubilization.

Structural proteins such as spider silk and silkworm silk are generally poorly soluble in aqueous and organic solutions, making them difficult to manipulate in manufacturing processes. Although some organic acids and alcohols, such as formic acid and hexafluoroisopropanol (HFIP), effectively solubilize poorly soluble proteins, little is known about their protein solubilization mechanism. In this study, the solubility of N-acetyl amino acid amide compounds in organic solvents-formic acid, acetic acid, HFIP and isopropanol-was measured to clarify the protein solubilization mechanism at the amino acid residue level. On the basis of thermodynamic analyses of the solubility in terms of the transfer free energy (from water to organic solvents), every organic solvent was found to be effective in thermodynamically stabilizing hydrophobic amino acid side chains in the liquid phase. Formic acid and HFIP were comparably effective in the stabilization of the polypeptide backbone, whereas acetic acid and isopropanol were ineffective. Therefore, the significant solubilizing effect of formic acid and HFIP on the structural proteins was attributed to their favorable interactions with hydrophobic amino acid side chains and with the polypeptide backbone of the proteins. The present findings are useful for the optimization of protein manipulation and amino acid sequence design.

A practical EPR dosimetry system for routine use in radiotherapy: uncertainty analysis of lithium formate dosimeters at the therapeutic dose level.

In electron paramagnetic resonance (EPR) dosimetry, solid dosimeter materials such as alanine (AL) or, more recently, lithium formate monohydrate (LFM) are typically used. These materials offer high potential for applications in radiotherapy based on their favorable dosimetric properties. Nevertheless, EPR dosimetry is not widespread in the clinics. This work presents an uncertainty analysis of EPR dosimetry in the dose range from 1 to 70 Gy using a compact spectrometer and applying a practical procedure being suitable for routine use in radiotherapy. The performances of self-pressed LFM pellets and commercial AL pellets are compared side by side. All pellets had a diameter of 4 mm and a height of 2 mm (AL) or 4 mm (LFM). The mean pellet mass was 35.81 mg and 73.81 mg for AL and LFM, respectively. Before irradiation, the pellets were stored for at least 8 weeks at 34 ± 2% relative humidity. For irradiation, the pellets were put inside an airtight capsule. In total, 25 pellets per material were examined. The pellets were irradiated at a temperature of 25 ± 2.5 (2σ) °C to doses of either 1, 5, 20, 50 or 70 Gy (five pellets per dose value and material) by a clinical 6 MV photon beam. Measurement uncertainties were obtained from five independent readouts per pellet within five weeks following irradiation using a benchtop EPR spectrometer. The measurement time of a single readout was restricted to 10 min per pellet. Dose values were derived from EPR signal amplitudes using a specifically developed spectral fitting procedure. Signal fading characteristics were analyzed and taken into account during evaluation. The relative dose uncertainties (1σ) for a single readout at doses ≥ 5 Gy are below 2.8% (AL) and 1.1% (LFM) but increase to 12.3% (AL) and 2.6% (LFM) at 1 Gy. By averaging five independent readouts, the uncertainties at 1 Gy decrease to 2.6% (AL) and 0.8% (LFM). In terms of dose uncertainty, the LFM pellets are superior to the commercial AL pellets owing to their narrower EPR spectrum and approximately doubled mass resulting in higher EPR signal intensities. In case of the LFM pellets, the EPR dosimetry system shows a high level of precision (< 3%) down to 1 Gy being preferable for applications in radiotherapy. The uncertainties can be further decreased by averaging multiple dose values from independent readouts.

Can a Nonorganometallic Ruthenium(II) Polypyridylamine Complex Catalyze Hydride Transfer? Mechanistic Insight from Solution Kinetics on the Reduction of Coenzyme NAD+ by Formate.

Application of organometallic ruthenium(II) arene complexes has been successful for the modulation of cellular redox processes via their interaction with species such as formate to control the NAD+/NADH balance in cells. Here we present the first evidence that similar effects can be reached with the application of a nonorganometallic ruthenium(II) polypyridyl complex. Kinetic studies performed demonstrate the ability of [RuII(terpy)(en)(H2O/EtOH)]2+ in water/ethanol (1:9, v/v) solution, where terpy = 2,2':6',2″-terpyridine and en = ethylenediamine, to catalyze the reduction of the NAD+ coenzyme to NADH in the presence of formate as hydride transfer source. In this case, terpy instead of arene is responsible for the labilization of coordinated solvent. The suggested catalytic cycle begins with the fast anation of the [RuII(terpy)(en)(H2O/EtOH)]2+ complex by formate. This is followed by the rate-determining formate-catalyzed decarboxylation of the generated ruthenium(II) formato complex to form [RuII(terpy)(en)H]+. Rapid hydride transfer to NAD+ from [RuII(terpy)(en)H]+ to form NADH and to regenerate the starting ruthenium(II) solvato complex, closes the overall catalytic cycle.

Color tuning of chlorophyll a and b pigments revealed from gas-phase spectroscopy.

Chlorophyll (Chl) pigments are responsible for vital mechanisms in photosynthetic proteins: light harvesting, energy transfer and charge separation. A complex interplay between the Chl molecule and its microenvironment determines its transition energy. Interactions such as excitonic coupling with one or more pigments (Chls or carotenoids), axial ligation to the magnesium center, or electrostatic interactions between Chl and nearby amino-acid residues all influence the photophysical properties. Here we use time-resolved photodissociation action spectroscopy to determine transition energies of Chla/b complexes in vacuo to directly compare the impact of a negatively charged axial ligand (formate) to that of exciton coupling between two Chls. Experiments carried out at the electrostatic ion storage ring ELISA allow dissociation to be sampled on hundreds of milliseconds time scale. Absorption-band maxima of Chla-formate complexes are found at 433 ± 4 nm/2.86 ± 0.03 eV (Soret band) and in the region 654-675 nm/1.84-1.90 eV (Q band) and those of Chla dimers tagged by a quaternary ammonium ion at 419 ± 5 nm/2.96 ± 0.04 eV (Soret band) and 647 nm/1.92 eV (Q band). The axial ligand strongly affects the Chla transition energies causing redshifts of 0.21 eV of the Soret band and 0.04-0.1 eV of the Q band compared to Chla tagged by a quaternary ammonium. Slightly smaller shifts were found in case of Chlb. The redshifts are approximately twice that induced by excitonic coupling between two Chlas, also tagged by a quaternary ammonium ion. Axial ligation brings the absorption by isolated Chls very close to that of photosynthetic proteins.

Method Validation of Acrylamide in Dried Blood Spot by Liquid Chromatography-tandem Mass Spectrometry.

BACKGROUND AND OBJECTIVE: Acrylamide (AA) is a carcinogenic substance that is easily found in working environment, food, contaminated air and tobacco smoke. This substance can be distributed rapidly through all body compartments. The aim of this study is to get the method for determining acrylamide in dried blood spot. MATERIALS AND METHODS: Dried blood spot was used as the bio-sampling method and was optimized and validated by using propranolol as the internal standard. The sample was prepared using a protein precipitation technique optimized. Reversed-phase chromatography with Acquity® UPLC BEH C18 column (1.7, 2.1× 100 mm) was used for compound separation. RESULTS: Optimized analytical condition for this substance was eluted with the flow rate of 0.20 mL/min under a gradient of the mobile phase of 0.1% formic acid in water and acetonitrile within 3 min. Triple quadrupole mass spectrometry with electrospray ionization (ESI) in positive mode was used as quantification analysis. The Multiple Reaction Monitoring (MRM) was set at m/z 71.99>55.23 (m/z) for acrylamide and 260.2>116.2 (m/z) for propranolol. The range of concentration was linear within 2.5-100 µg mL-1. CONCLUSION: All the validation parameters were fulfilled the criteria in US FDA Guideline for Bioanalytical Method Validation 2018.

Catalytic Transfer Hydrogenation of Low-erucic-acid Rapeseed Oil over a Ni-Ag0.15/SBA15 Catalyst.

The kinetics of catalytic transfer hydrogenation (CTH) of low-erucic-acid rapeseed oil using ammonium formate as a hydrogen donor over a Ni-Ag0.15/SBA15 catalyst were studied. Then, a kinetic model for the hydrogenation of low-erucic-acid rapeseed oil was established, and it was found that the reaction rate constants of hydrogenations of 9c-18:1 and 12c-18:1 oleic acid were 0.1262 and 0.0148, and the catalytic selectivity of linoleic acid was 2.04. For the catalyst loading of 0.23%, the hydrogenation temperature was 80°C, the ammonium formate concentration was 0.32 mol/50 mL, and the low-erucic-acid rapeseed oil was hydrogenated in 90 min; it was also found that the iodine value of low-erucic-acid rapeseed oil was 80 g I2/100 g, the oleic acid content was 65%, and the trans fatty acids (TFAs) content was only 6.7%. Therefore, CTH may be widely used in the modification of oils and fats.

Peptide separation selectivity in proteomics LC-MS experiments: Comparison of formic and mixed formic/heptafluorobutyric acids ion-pairing modifiers.

Separation selectivity and detection sensitivity of reversed-phase high-performance liquid chromatography with tandem mass spectrometry analyses were compared for formic (0.1%) and formic/heptafluorobutyric (0.1%/0.005%) acid based eluents using a proteomic data set of ∼12 000 paired peptides. The addition of a small amount of hydrophobic heptafluorobutyric acid ion-pairing modifier increased peptide retention by up to 10% acetonitrile depending on peptide charge, size, and hydrophobicity. Retention increase was greatest for peptides that were short, highly charged, and hydrophilic. There was an ∼3.75-fold reduction in MS signal observed across the whole population of peptides following the addition of heptafluorobutyric acid. This resulted in ∼36% and ∼21% reduction of detected proteins and unique peptides for the whole cell lysate digests, respectively. We also confirmed that the separation selectivity of the formic/heptafluorobutyric acid system was very similar to the commonly used conditions of 0.1% trifluoroacetic acid, and developed a new version of the Sequence-Specific Retention calculator model for the formic/heptafluorobutyric acid system showing the same ∼0.98 R2 -value accuracy as the Sequence-Specific Retention calculator formic acid model. In silico simulation of peptide distribution in separation space showed that the addition of 0.005% heptafluorobutyric acid to the 0.1% formic acid system increased potential proteome coverage by ∼11% of detectable species (tryptic peptides ≥ four amino acids).

Improved Annotation of Untargeted Metabolomics Data through Buffer Modifications That Shift Adduct Mass and Intensity.

Annotation of untargeted high-resolution full-scan LC-MS metabolomics data remains challenging due to individual metabolites generating multiple LC-MS peaks arising from isotopes, adducts, and fragments. Adduct annotation is a particular challenge, as the same mass difference between peaks can arise from adduct formation, fragmentation, or different biological species. To address this, here we describe a buffer modification workflow (BMW) in which the same sample is run by LC-MS in both liquid chromatography solvent with 14NH3-acetate buffer and in solvent with the buffer modified with 15NH3-formate. Buffer switching results in characteristic mass and signal intensity changes for adduct peaks, facilitating their annotation. This relatively simple and convenient chromatography modification annotated yeast metabolomics data with similar effectiveness to growing the yeast in isotope-labeled media. Application to mouse liver data annotated both known metabolite and known adduct peaks with 95% accuracy. Overall, it identified 26% of ∼27 000 liver LC-MS features as putative metabolites, of which ∼2600 showed HMDB or KEGG database formula match. This workflow is well suited to biological samples that cannot be readily isotope labeled, including plants, mammalian tissues, and tumors.

From Extra Virgin Olive Oil to Refined Products: Intensity and Balance Shifts of the Volatile Compounds versus Odor.

To explore relationships between the volatile organic compounds (VOCs) of different grades of olive oils (OOs) (extra virgin olive oil (EVOO), refined olive oil (ROO), and pomace olive oil (POO)) and odor quality, VOCs were measured in the headspace of the oils by proton transfer reaction quadrupole ion guide time-of-flight mass spectrometry. The concentrations of most VOCs differed significantly between the grades (EVOO>ROO>POO), whereas the abundance of m/z 47.012 (formic acid), m/z 49.016 (fragments), m/z 49.027 (fragments), and m/z 115.111 (heptanal/heptanone) increased in that order. Although the refined oils had considerably lower VOC abundance, the extent of the decline varied with the VOCs. This results in differences in VOCs proportions. The high VOC abundance in the EVOO headspace in comparison to ROO and POO results in a richer and more complex odor. The identified C5-C6 compounds are expected to contribute mainly to the green odor notes, while the identified C1-C4 and C7-C15 are mainly responsible for odor defects of OOs. Current results reveal that processing strongly affects both the quantitative and relative abundance of the VOCs and, therefore, the odor quality of the various grades of OOs.