Early sponges and toxic protists: possible sources of cryostane,an age diagnostic biomarker antedating Sturtian Snowball Earth

The period 800–717 million years (Ma) ago, in the lead‐up to the Sturtian Snowball glaciation, saw an increase in the diversity of eukaryotic microfossils. To afford an independent and complementary view of this evolutionary period, this study presents the distribution of eukaryotic biomarkers from three pre‐Sturtian successions across the supercontinent Rodinia: the ca. 780 Ma Kanpa Formation of the Western Australian Officer Basin, the ca. 800–740 Ma Visingsö Group of Sweden, and the 740 Ma Chuar Group in Arizona, USA. The distribution of eukaryotic steranes is remarkably similar in the three successions but distinct from all other known younger and older sterane assemblages. Cholestane was the only conventional structure, while indigenous steranes alkylated in position C‐24, such as ergostane, stigmastane, dinosterane and isopropylcholestane, and n‐propylcholestane, were not observed. This sterane distribution appears to be age diagnostic for the pre‐Sturtian Neoproterozoic. It attests to the distinct evolutionary state of pre‐Snowball eukaryotes, pointing to a taxonomic disparity that was still lower than in the Ediacaran (635–541 Ma). All three basins also show the presence of a new C₂₈ sterane that was tentatively identified as 26‐methylcholestane, here named cryostane. The only known extant organisms that can methylate sterols in the 26‐position are demosponges. This assignment is plausible as molecular clocks place the appearance of the earliest animals into the pre‐Sturtian Neoproterozoic. The unusual 26‐methylsterol may have protected sponges, but also other eukaryotes, against their own membranolytic toxins. Some protists release lytic toxins to deter predators and kill eukaryotic prey. As conventional membrane sterols can be the site of attack for these toxins, sterols with unusual side‐chain modification protect the cell. This interpretation of cryostane supports fossil evidence of predation in the Chuar Group and promotes hypotheses about the proliferation of eukaryophagy in the lead‐up to the Cryogenian.     

The “Dirty Ice” of the McMurdo Ice Shelf: Analogues for biological oases during the Cryogenian

The Cryogenian (~717–636 Ma) is characterized by widespread glaciation and dramatic fluctuations in biogeochemical cycling during the Sturtian and Marinoan glaciations. The Snowball Earth hypothesis posits that during this period, ice‐covered oceans of more or less global extent shut down or greatly diminished photosynthesis in the marine realm. However, rather than suffering a catastrophic loss of biodiversity, fossil evidence suggests that major eukaryotic lineages survived and, indeed, the end of the Cryogenian marks the onset of a rapid diversification of eukaryotic life. Persistence of diverse life forms through glaciations is thought to have occurred in supraglacial refugia although the exact nature and full extent of such habitats remain uncertain. We present further evidence for the diversity and characteristics of supraglacial ecosystems on the McMurdo Ice Shelf in Antarctica and suggest that refugia analogous to “dirty ice,” that is debris‐covered ice shelf ecosystems, potentially provided nutrient‐rich and long‐lasting biological Cryogenian oases. We also discuss how features of the McMurdo Ice Shelf indicate that mechanisms exist whereby material can be exchanged between the shallow sea floor and the surfaces of ice shelves along continental margins, providing vectors whereby ice shelf ecosystems can nourish underlying seafloor communities and vice versa.     

Paleogene stable isotope events

The oxygen isotope record in Paleogene benthic Foraminifera shows that at the base of the Paleogene the ocean deep waters had a temperature of about 10°C, rising to about 12°C at the base of the Eocene and cooling between 51 Ma and 49 Ma to about 9°C. The most dramatic event occurred just after the Eocene/Oligocene boundary, at about 35.8 Ma, when ocean deep waters cooled by several degrees within 10⁴–10⁵ yr, probably in association with temporary glaciation in the Antarctic region. Another more intense glacial event in Antarctica may have occurred later in the Oligocene, at about 31 Ma and a third near the top of the Oligocene at 24 Ma.In the marine carbon isotope record a very rapid negative excursion occurred precisely at the Cretaceous/Tertiary boundary. A recovery to unusually positive values in the Late Paleocene was followed by a second negative excursion close to the Paleocene/Eocene boundary that was even more extreme in magnitude although it was not as rapid. These major carbon isotope events permit very accurate stratigraphic correlation; there are many other smaller features in the carbon isotope record that will also prove useful for this purpose.     

Tectonic regime transition of the western South China Block in early Cambrian: Evidence from the Meishucun volcanic ash beds

The tectonic evolution of the South China Block, especially its western part, is ambiguous in the early Paleozoic owing to the lack of contemporary tectono-magmatic records. Thick ash beds preserved in the early Cambrian successions in the eastern Yunnan Province of the western South China Block provide new insights into their magmatic origins and tectonic evolution. We report here integrated in-situ zircon U-Pb data, Hf-O isotopes, and whole-rock geochemistry of two early Cambrian ash beds (Bed 5 and Bed 9) from the Meishucun section in the eastern Yunnan Province. SIMS zircon U-Pb data indicate the two ash layers were deposited at 533.2 ± 3.8 Ma and 526.2 ± 4.1 Ma, respectively. Zircon Hf-O isotopes and whole-rock geochemistry of the two ash beds demonstrate crustal origins of their parental magma and their close affinities to A-type granites. These features suggest that the ash beds were formed in extensional settings and the tectonic regime of the study area switched from compression to extension in the early Cambrian. The Meishucun volcanic records, in combination with other late Ediacaran–early Paleozoic magmatism along the northern margin of East Gondwana, may represent the initial opening phase of the Paleo-Tethys Ocean.     

Trace determination and isotopic analysis of the elements in life sciences by mass spectrometry

The main ionization methods in a mass spectrometer for isotope ratio determinations of the elements are discussed in this review. These methods are thermal ionization, spark source, electron impact, inductively coupled plasma and field desorption. As concerns thermal ionization, electron impact and field desorption, a survey of the possibilities of isotope analyses in the periodic table of the elements is given. Besides kinetic studies, trace element determination by isotope dilution technique is the main application for isotope ratio measurements of the elements. The definitive method, isotope dilution mass spectrometry, is discussed as a potential tool for achieving accurate and precise trace analyses. Using field desorption mass spectrometry, one example of calcium kinetics in man and one example of thallium trace determination in an animal tissue are given. Other metal trace analyses with the isotope dilution technique are presented for biological and medical samples using positive thermal ionization mass spectrometry. Negative thermal ions are formed for the mass spectrometric analysis of non-metals and non-metal compounds in food samples, e.g. for iodine and nitrate in milk powder. Preliminary results with the isotope dilution technique are presented for a new quadrupole thermal ionization mass spectrometer which is a low-cost instrument and can be easily handled.     

山西三叠系二马营组和铜川组SHRIMP锆石铀-铅年龄及其地质意义(英文)

采集了山西省二马营组和铜川组共4个层位的火山凝灰岩,在高灵敏度高分辨率二次离子质谱仪上以铀-铅法测定其中锆石的年龄。二马营组上部的样品测年结果为245.9 Ma±3.2 Ma, 铜川组一段样品测年结果为243.1 Ma±3.9 Ma, 二段上部两个样品年龄为238.6 Ma±2.6 Ma和234.6 Ma±6.5 Ma。结果表明出产陕北肯氏兽动物群的二马营组下部极有可能属于下三叠统,而铜川组时代可能从安尼期晚期到拉丁期。     

Facile preparation of deuterium-labeled N-acylhomoserine lactones as internal standards for isotope dilution mass spectrometry

N-Acylhomoserine lactones (AHLs) are widely conserved signal molecules that mediate quorum sensing in Gram-negative bacteria. In this study, deuterium-labeled AHLs were prepared for use as internal standards for isotope dilution mass spectrometry. Their utility in the sensitive and precise quantification of AHLs in culture supernatants of bacteria by GC/MS was demonstrated.     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.     

Sulphur cycling in a Neoarchaean microbial mat

Multiple sulphur (S) isotope ratios are powerful proxies to understand the complexity of S biogeochemical cycling through Deep Time. The disappearance of a sulphur mass‐independent fractionation (S‐MIF) signal in rocks <~2.4 Ga has been used to date a dramatic rise in atmospheric oxygen levels. However, intricacies of the S‐cycle before the Great Oxidation Event remain poorly understood. For example, the isotope composition of coeval atmospherically derived sulphur species is still debated. Furthermore, variation in Archaean pyrite δ³⁴S values has been widely attributed to microbial sulphate reduction (MSR). While petrographic evidence for Archaean early‐diagenetic pyrite formation is common, textural evidence for the presence and distribution of MSR remains enigmatic. We combined detailed petrographic and in situ, high‐resolution multiple S‐isotope studies (δ³⁴S and Δ³³S) using secondary ion mass spectrometry (SIMS) to document the S‐isotope signatures of exceptionally well‐preserved, pyritised microbialites in shales from the ~2.65‐Ga Lokammona Formation, Ghaap Group, South Africa. The presence of MSR in this Neoarchaean microbial mat is supported by typical biogenic textures including wavy crinkled laminae, and early‐diagenetic pyrite containing <26‰ μm‐scale variations in δ³⁴S and Δ³³S = ?0.21 ± 0.65‰ (±1σ). These large variations in δ³⁴S values suggest Rayleigh distillation of a limited sulphate pool during high rates of MSR. Furthermore, we identified a second, morphologically distinct pyrite phase that precipitated after lithification, with δ³⁴S = 8.36 ± 1.16‰ and Δ³³S = 5.54 ± 1.53‰ (±1σ). We propose that the S‐MIF signature of this secondary pyrite does not reflect contemporaneous atmospheric processes at the time of deposition; instead, it formed by the influx of later‐stage sulphur‐bearing fluids containing an inherited atmospheric S‐MIF signal and/or from magnetic isotope effects during thermochemical sulphate reduction. These insights highlight the complementary nature of petrography and SIMS studies to resolve multigenerational pyrite formation pathways in the geological record.     

Large-scale total synthesis of <Superscript>13</Superscript>C<Subscript>3</Subscript>-labeled citrinin and its metabolite dihydrocitrinone

The analysis of the nephrotoxic mycotoxin citrinin in food, feed, and physiological samples is still challenging. Nowadays, liquid chromatography coupled with mass spectrometry is the method of choice for achieving low limits of detection. But matrix effects can present impairments for this method. Stable isotope dilution analysis can prevent some of these problems. Therefore, a stable isotopically labeled standard of citrinin for use in stable isotope dilution analysis was synthesized on large scale. The improved diastereoselective total synthetic strategy offered the possibility to introduce three ¹³C-labels in two steps by ortho-toluate anion chemistry. This led to a mass difference of 3 Da, sufficient for preventing spectral overlap. Additionally, a stable isotopically labeled form of dihydrocitrinone, the main urinary metabolite of citrinin, was synthesized with the same mass difference. This was achieved by a sequence of cyclisation, oxidation, deprotection, and carboxylation reactions starting from a protected intermediate of the labeled citrinin synthesis. Thus, this method also offers a complete way to synthesize dihydrocitrinone from citrinin on large scale.     

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high‐abundant serum proteins by partial denaturation and enrichment of low‐abundant biomarkers by size exclusion chromatography. The recovery of low‐abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody‐based strategies, and offers the possibility of multiplexing. Our proof‐of‐principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.     

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, ''hook-effect'').¹ An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).² In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules ^{3, 4} and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.^5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.^8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Effect of different doses of organic fertilizer (cow dung) on pond productivity and fish biomass in stillwater ponds

The effects of five (5 000, 10 000, 15 000, 20 000, 24 000 kg ha^?1 year^?1) different doses of organic fertilizer (cow dung) were studied on pond productivity in terms of plankton production and fish biomass in freshwater fish ponds. The grow out period was 60 days. Physico-chemical factors of pond waters were also monitored. With an increase in the fertilizer dose, biochemical oxygen demand (BOD) (1.7 ± 0.1 – 10.35 ± 0.05 mg L^?1), O-PO₄ (0.04 ± 0.0 – 0.77 ± 0.02 mg L^?1) and NH₄-N (0.03 ± 0.02 – 0.32 ± 0.02 mg L^?1) increased significantly (P < 0.05). Alkalinity (79.0 ± 1.6 – 164.0 ± 3.8 mg L^?1) also increased with the increase in fertilizer dose, declining after 60 and 75 days (48.8 ± 1.13 – 67.9 ± 2.1 mg L^?1). NO₃-N was maximum (1.66 ± 0.2 mg L^?1) in the ponds which received cow dung at 15 000 kg ha^?1 year^?1, and declined (0.94 ± 0.5 mg L^?1) at higher doses. Dissolved oxygen (DO) remained significantly high (4.7 mg L^?1) up to the third (15 000 kg ha^?1 year^?1) treatment. Highest plankton population (phytoplankton 17 350.0 ± 1 250.0 no L^?1), zooplankton (373.0 ± 22.0 no L^?1), species diversity (phytoplankton 3.0, zooplankton 2.3), fish biomass (4.45 kg) and specific growth rate (SGR) (2.36 % body weight (BW) d^?1) were also observed in ponds which were treated with fertilizer at 15 000 kg ha^?1 year^?1. However, at higher doses, a decline in these parameters (phytoplankton, 0.0 – 8 810.0 ± 690.0 no L^?1; zooplankton, 0.0 – 205.0 ± 25.0 no L^?1; fish biomass, 2.3 kg; SGR, 1.25 % body weight (BW) d^?1) was observed. Furthermore, with a decrease in the water temperature from 24 °C (on day 60) to 21 °C (on day 75), a decline in nutrient release, plankton population L^?1 and species diversity was observed. Sediment analysis indicated that with an increase in the fertilizer dosage, a significant and progressive increase in the accumulation of organic carbon (0.787 ± 0.006 – 0.935 ± 0.01), total nitrogen (0.877 ± 0.071 – 1.231 ± 0.03), NH₄-N (54.4 ± 0.57 – 68.95 ± 0.81), NO₃-N (78.5 ± 1.21 – 98.5 ± 0.35), total P (140.0 ± 0.50 – 151.0 ± 1.27) and soluble P (7.15 ± 0.18 – 10.1 ± 0.56) took place; similarly, electrical conductivity (EC) values of sediment also increased progressively (from 200.0 ± 7.1–300.0 ± 10.63 μ mhos cm^?1).     

Carbon isotope ratio (δC) values of urinary steroids for doping control in sport

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (δ¹³C) profiling study (n = 1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This δ¹³C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to δ¹³C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n = 100; consisting of three countries) is also presented with comparison to δ¹³C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.     

Prostacyclin is not a circulating hormone in man

A highly specific stabel isotope dilution assay for plasma 6-oxo-prostaglandin F_1α has been developed. The method employs capillary column gas chromatography coupled with negative ion chemical ionisation mass spectrometry. The limit of sensitivity of the assay is 0.5 pg.ml⁻¹. Concentrations of 6-oxo-prostaglandin F_1α in the plasma of 20 healthy volunteers determined by this assay were all below 3 pg.ml⁻¹. The levels were much lower than any previously reported and confirms that prostacyclin is not a circulating hormone in man under normal physiological conditions.     

Using stable isotope ratios as a tracer of feeding adaptation in released Japanese flounder Paralichthys olivaceus

Release‐recapture experiments were conducted to examine temporal changes of the carbon and nitrogen stable isotope (δ¹³C and δ¹⁵N) ratios in the muscle tissue of artificially produced Japanese flounder Paralichthys olivaceus, juveniles. About 9000 juveniles (mean ± s .d . 43·3 ± 5·2 mm in standard length and 1·07 ± 0·37 g, n = 15) were released in each of three coastal areas: Chojagasaki, Arasaki and Jogashima with different geographical conditions, along Sagami Bay, Pacific coast of central Japan. Recapture efforts were made on 4, 11, 18, 40 and 55 days after the release. The stable isotope ratios, RNA:DNA ratio, stomach content mass (per body mass M_sc) and condition factor (K) of recaptured individuals were measured. The mean ± s .d . δ¹³C and δ¹⁵N values (n = 15) were ?18·3 ± 0·2‰ and 12·2 ± 0·2‰, respectively at the release. Wild Japanese flounder juveniles were captured only in Chojagasaki, and the δ¹³C and δ¹⁵N values (n = 6) were ?14·0 ± 0·4‰ and 13·2 ± 0·7‰, respectively; these values were considered to represent the wild diet. Nutritional conditions of the released and recaptured juveniles as determined by the RNA : DNA ratio, M_SC and K were indicated to be the best in Chojagasaki, in which the stable isotope ratios gradually shifted towards and reached the wild values within 40 days. This result along with stomach content analyses suggested that the released juveniles had acquired a wild feeding habit. In Arasaki and Jogashima, nutritional conditions of the recaptured juveniles were poorer, with no clear changes in the stable isotope ratios. Greatly varied stable isotope ratio values were observed in the juveniles recaptured in Chojagasaki 11 days after the release, ranging from the release levels to the wild levels. The extent of changes in the stable isotope ratios had a positive correlation to the RNA : DNA ratio and K of these juveniles (r = 0·87, n = 10 and r = 0·83, n = 18, respectively). The analyses of stable isotope ratios coupled with nutritional condition were considered to be an effective tool to examine post‐release feeding adaptation of Japanese flounder juveniles.     

Quantitative analysis of two dinor urinary metabolites of prostaglandin I2

The synthesis of deuterium- and tritium-labeled analogs of 2,3-dinor-6-keto-prostaglandin F_1α and of 6,15-diketo-13,14-dihydro-2,3-dinor-prostaglandin F_1α is described. These analogs were used as internal standards in the assay of the corresponding unlabeled metabolites in human urine by stable isotope dilution and combined gas chromatography-mass spectrometry. In male subjects the 24-h urinary excretion of the two metabolites was found to be 719 ± 264 and 314 ± 115 ng, respectively. The method offers a noninvasive approach to the study of prostaglandin I₂ synthesis in man.     

Improved in vivo measurement of alternative oxidase respiration in field‐collected pine roots

Cellular respiration via the alternative oxidase pathway (AOP) leads to a considerable loss in efficiency. Compared to the cytochrome pathway (COP), AOP produces 0–50% as much ATP per carbon (C) respired. Relative partitioning between the pathways can be measured in vivo based on their differing isotopic discriminations against ¹⁸O in O₂. Starting from published methods, we have refined and tested a new protocol to improve measurement precision and efficiency. The refinements detect an effect of tissue water content (P < 0.0001), which we have removed, and yield precise discrimination endpoints in the presence of pathway‐specific respiratory inhibitors [CN^? and salicylhydroxamic acid (SHAM)], which improves estimates of AOP/COP partitioning. Fresh roots of Pinus sylvestris were sealed in vials with a CO₂ trap. The air was replaced to ensure identical starting conditions. Headspace air was repeatedly sampled and isotopically analyzed using isotope‐ratio mass spectrometry. The method allows high‐precision measurement of the discrimination against ¹⁸O in O₂ because of repeated measurements of the same incubation vial. COP and AOP respiration discriminated against ¹⁸O by 15.1 ± 0.3‰ and 23.8 ± 0.4‰, respectively. AOP contributed to root respiration by 23 ± 0.2% of the total in an unfertilized stand. In a second, nitrogen‐fertilized, stand AOP contribution was only 14 ± 0.2% of the total. These results suggest the improved method can be used to assess the relative importance of COP and AOP activities in ecosystems, potentially yielding information on the role of each pathway for the carbon use efficiency of organisms.