Isotope Ratio Measurements of Iron in Blood Samples by Multi-collector ICP-MS to Support Nutritional Investigations in Humans

With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., ⁵⁴Cr⁺ on ⁵⁴Fe⁺ and ⁵⁸Ni⁺ on ⁵⁸Fe⁺) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008–0.05 (% RSD), 0.007–0.015 (% RSD), and 0.03–0.09 (% RSD) were obtained for ⁵⁴Fe/⁵⁶Fe, ⁵⁷Fe/⁵⁶Fe, and ⁵⁸Fe/⁵⁶Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.     

Absorption of non-haem iron from food during normal pregnancy.

OBJECTIVE--To determine whether the increased iron demands of pregnancy could be met by increased absorption from dietary sources. DESIGN AND SETTING--Longitudinal prospective study in the research unit of a maternity hospital. SUBJECTS--12 normal pregnant women. INTERVENTIONS--At 12, 24, and 36 weeks'' gestation (within one week) and 16-24 weeks after delivery women ate a breakfast of meat, bread, and orange juice (3.2 mg iron), extrinsically labelled with the stable isotope iron-54 (2.8 mg); the stable isotope iron-57 (200 micrograms) was given intravenously. MAIN OUTCOME MEASURES--Serum samples were taken for 10 hours after administration of the isotopes; ratios of the isotopes were measured by inductively coupled plasma mass spectrometry, and the absorption of oral iron was calculated. RESULTS--The geometric mean (95% confidence interval) absorption of iron at 12, 24, and 36 weeks'' gestation was 7% (5% to 11%), 36% (28% to 47%), and 66% (57% to 76%) respectively. At 16-24 weeks after delivery the absorption was 11% (6% to 21%). The mean increase in absorption at 36 weeks (compared with that at 12 weeks) was 9.1 times (6.0 to 13.7). One pregnant woman developed iron deficiency anaemia but was otherwise indistinguishable from the others. CONCLUSIONS--An increase in the absorption of iron from food is a physiological consequence of normal pregnancy, not the result of developing anaemia during pregnancy, and such an increase is large enough to meet the increased requirements of pregnancy provided that the dietary intake is adequate.     

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.     

Iron balance in the common vampire bat Desmodus rotundus

1. Total body iron in the common vampire bat was 80 +/- 67 mg Fe/kg body wt (mean +/- SD). 2. Mean (+/- SD) iron absorption as measured by the double isotope method was 0.068 +/- 0.0032% Fe. A typical adult bat ingests 6.1 mg Fe/day (Morton & Wimsatt, 1980) of which approximately 4.2 microgram Fe is absorbed. 3. Body iron turnover (BIT) was estimated from the decrease in specific radioactivity of the blood over more than a year. The mean (+/- SD) of the half-life of iron turnover was 379 +/- 101 days and that of the estimate of BIT. 0.14 +/- 0.04% TBI/day. This is equivalent to a loss of approximately 4.6 microgram Fe/day. 4. The common vampire bat maintains iron balance by severely limiting the percentage of iron absorbed from its very high iron diet.     

Benthic iron cycling in a high‐oxygen environment: Implications for interpreting the Archean sedimentary iron isotope record

The most notable trend in the sedimentary iron isotope record is a shift at the end of the Archean from highly variable δ⁵⁶Fe values with large negative excursions to less variable δ⁵⁶Fe values with more limited negative values. The mechanistic explanation behind this trend has been extensively debated, with two main competing hypotheses: (i) a shift in marine redox conditions and the transition to quantitative iron oxidation; and (ii) a decrease in the signature of microbial iron reduction in the sedimentary record because of increased bacterial sulfate reduction (BSR). Here, we provide new insights into this debate and attempt to assess these two hypotheses by analyzing the iron isotope composition of siderite concretions from the Carboniferous Mazon Creek fossil site. These concretions precipitated in an environment with water column oxygenation, extensive sediment pile dissimilatory iron reduction (DIR) but limited bacterial sulfate reduction (BSR). Most of the concretions have slightly positive iron isotope values, with a mean of 0.15‰ and limited iron isotope variability compared to the Archean sedimentary record. This limited variability in an environment with high DIR and low BSR suggests that these conditions alone are insufficient to explain Archean iron isotope compositions. Therefore, these results support the idea that the unusually variable and negative iron isotope values in the Archean are due to dissimilatory iron reduction (DIR) coupled with extensive water column iron cycling.     

Combined fluorescence histochemistry and 3h-noradrenaline measurements of adrenergic nerves

Summary A method is described which combines the histochemical fluorescence technique of Falck and Hillarp with isotope measurements in the same pieces of tissue. Tissue pieces incubated in isotope solutions were treated for fluorescence microscopy and examined. They were then removed from the microscopical slides, and the radioactivity determined. It was shown that NA¹ content and estimated fluorescence intensity were well correlated. The procedure devised is of special value when isotope measurements are needed of structures which can be safely identified only in the fluorescence microscope, and it has been used for quantitative estimations of adrenergic innervation.Abbreviations used Na noradrenaline - cpm counts per minute - dpm desintegrations per minute     

Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the alpha-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.     

Secondary 15N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases

Secondary 15N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these 15N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer [Cook, P. F., Oppenheimer, N. J. & Cleland, W. W. (1981) Biochemistry 20, 1817]. If this mechanism were valid, as N-1 becomes pyramidal an 15N isotope effect of up to 2-3% would be observed. In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant 15N kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.     

Comparison of faecal monitoring and area under the curve techniques to determine iron absorption in humans using stable isotope labelling

The objective of the study was to compare faecal monitoring and area under the curve (AUC) techniques for measuring iron absorption from a stable isotope label. Nine healthy female subjects (age: 33±8 yr) were recruited to take part in the study. After an overnight fast, each subject received a 5 mg dose of highly enriched Fe-57 and all faecal samples were collected for 10 days post-dose to allow for estimation of iron absorption by the faecal monitoring method (geometric mean: 25%). Serial blood samples (12 in total) were also collected from each volunteer for 6 h post-dose to estimate iron absorption by the well-validated area under the plasma iron concentration curve (AUC) method (geometric mean: 18%). The faecal monitoring method yields higher iron absorption results compared to AUC. This discrepancy may be due to the steps involved in faecal processing where systematic errors are likely to take place and also to the lack of complete faecal recovery associated with issues of cleanliness and hygiene when passing stools. Both of these lead to losses of the unabsorbed isotope and to an overestimate of iron absorption.     

Studies on the role of iron in zinc toxicity in chicks

The interaction of dietary iron and zinc was studied in chicks. Zinc was found to be more toxic in iron-deficient animals than iron-supplemented animals as measured by hemoglobin concentrations and growth. Analyses of the kidney and liver for iron and zinc were carried out. As the level of iron was increased from 0-1000 ppm supplementation, the concentration of liver zinc increased. The organ levels of iron were decreased as the dietary zinc levels were increased from 0-5000 ppm. Radioisotope studies using⁶⁵Zn revealed that the iron content of the diet did not affect absorption of zinc. Administration of the isotope, either in an intestinal segment or intravenously, resulted in more zinc being taken up by the liver in the iron supplemented animals. This was especially noted when the ratio of the isotope in liver to that in the blood was compared. Gel chromatography of kidney and liver homogenates revealed that iron deficiency resulted in less zinc being eluted in a volume characteristic of metallothionein compared to homogenates of organs from iron supplemented animals. The results indicate that iron-supplemented animals have a greater capacity for sequestering zinc on metallothionein than do iron-deficient animals. Conversely, iron-deficient chicks were more susceptible to the effects of zinc toxicity than are iron-adequate chicks.     

Determination of deoxycholic acid pool size and input rate using [24-13C]deoxycholic acid and serum sampling

We have developed an isotope dilution method for determination of deoxycholic acid pool size and input rate which employs oral administration of 50 mg of [24-13C]deoxycholic acid and serum sampling. The method has been validated by classical isotope dilution technique using [24-14C]deoxycholic acid and bile sampling in five patients with colonic adenomas. Excellent agreement between pool sizes and input rates determined with 13C/12C isotope ratio measurements in serum and 14C measurements in bile was obtained when isotope ratios were measured in the conjugated fraction of deoxycholic acid in serum. We conclude that pool size and input rate of deoxycholic acid can accurately be determined by blood sampling after oral administration of [24-13C]deoxycholic acid, therewith eliminating the use of radioactive tracers and the need for bile sampling.     

Measurement of isotope ratios in organic compounds at picomole quantities by capillary gas chromatography/quadrupole electron impact mass spectrometry

Measurements of isotope ratios in organic compounds at nanomolar concentrations in biological fluids require sensitive and selective gas chromatographic/mass spectrometric methods. The efficiency of sample preparation procedures must be high and gas chromatographic conditions must assure high resolution chromatography producing narrow, intense peaks. Mass spectrometric conditions must create suitable mass fragments, preferably in the high mass range. Detection, controlled by selected ion monitoring (SIM), requires the use of small mass intervals and adequate acquisition times. However, the choice of optimal conditions is limited by the need to acquire multiple data points for the eluting compound. The effects of these variables on the precision of isotope ratio measurements have been investigated to develop an optimal method for isotope ratio measurements in serum bile acids at low concentrations (0.05-8 micromol l-1). With a 25 m X 0.32 mm fused silica capillary OV-1701 column and cold on-column injection, peak widths of 10 s were obtained. Quadrupole mass spectrometry in the electron impact ionization mode (70 eV) and selected ion monitoring (SIM)(mass interval 1/16 u, acquisition time 100 ms) allowed precise (cv less than 1.5%) isotope ratio measurements for chenodeoxycholic, cholic and deoxycholic acid in a single run at quantities as low as 5 pmol injected on the column. SIM switching during the run permitted isotope ratio measurements (cv less than 2.2%) for an additional six bile acids, normally detected in the serum of healthy subjects.     

Results of an international round robin for the quantification of serum non-transferrin-bound iron: Need for defining standardization and a clinically relevant isoform

Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n=11) and secondary (n=1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods differed widely in mean serum NTBI level (range 0.12-4.32mumol/L), between-sample variation (SD range 0.20-2.13mumol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45mumol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated significantly (R(2) range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related differently from those of serum transferrin saturation (TS) when expressed in terms of both regression coefficients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes.     

Reliability in estimates of body composition of birds: oxygen-18 versus deuterium dilution

Body composition in birds was evaluated indirectly by 18O and 2H dilution. Body composition was determined by whole-body chemical analysis of eight adult roosters (Gallus gallus). In vivo measurements of total body water (TBW) were carried out using doubly labeled water (2H2 18O). Estimated dilution spaces using both the plateau and intercept approaches were compared with the results obtained by carcass lyophilization. Both 18O and 2H slightly overestimated TBW compared with the results obtained by lyophilization, by 2.2%+/-1.9% and 5.7%+/-0.2%, respectively; both differences were statistically significant (P<0.01). The difference between these isotope estimations was significant (P<0.001). However, isotope dilution spaces and TBW were highly correlated. There was a strong inverse relationship between total body fat and TBW percentages (r2=0.98, P<0.0001). The relation between TBW and body protein was significant. Water content in lean body mass (72.8%) obtained in our study was very close to that reported in mammals, demonstrating no fundamental difference in tissue water content between birds and mammals. Estimated body fat and protein values from isotopic dilution did not significantly differ from values obtained by direct chemical analysis (P>0.05), except for body fat in the Pace and Rathbun approach (Table 3). Although estimation of TBW and body composition by isotope dilution is time consuming and expensive, deuterium offers a reliable and low-cost alternative compared with 18O. The advantage of in vivo estimation of TBW with isotopic dilution in combination with the regression approach is that it permits repeated measurements of body composition on the same birds under laboratory and free-living conditions.     

Superoxide ion as a primary reductant in ascorbate-mediated ferritin iron release

The mechanism of ascorbate-promoted ferritin iron reduction under aerobic conditions was studied. The initial rate of ferritin iron release was determined by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex which absorbs at 562 nm. Variation of the initial ferrozine concentration had no influence on the rate of iron release suggesting that ferrozine does not participate in the rate-determining step. Experimental measurements of the initial rate of iron release as a function of ascorbate concentration resulted in saturation kinetics with Vmax = 2.0 X 10(-7) M.min-1 and KM = 1.3 X 10(-3) M. The effect of pH was quite pronounced with a maximal rate of iron release at pH 7.0. Stoichiometric measurements on the reaction mixture, with added catalase, resulted in a ratio of 2 Fe(II) released per ascorbate. Ascorbate-mediated iron release was inhibited 85% by superoxide dismutase, but 0% inhibition was noted with aposuperoxide dismutase. It is proposed that superoxide ion, generated during the iron-promoted oxidation of ascorbate, acts as a reductant of ferritin iron. A mechanism of ferritin iron release consistent with these experimental observations is discussed.     

A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction

The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.     

Siderophore iron transport followed by electron paramagnetic resonance spectroscopy

Siderophore iron transport was followed in Ustilago sphaerogena using isotope transport assays coupled with EPR spectroscopy. EPR spectroscopy was used as a quantitative tool to follow the rate of reduction of siderophore iron(III) to iron(II) in the cell suspension by following the disappearance of the signal at g = 4.3. This rate was compared with the rate of iron transport, measured by the disappearance of radioactively labeled iron from the medium. The transport of three iron chelates was examined: the ferric siderophores ferrichrome and ferichrome A, and iron(III) chelated to excess citrate. For the transport of ferrichrome, an iron(III) ionophore, the rate of reduction of iron(III) to iron(II) was significantly lower than the rate of uptake of isotope from the medium supernatant, which is consistent with the established mechanism of uptake of the entire complex followed by intracellular reduction to remove the iron from the ligand. However, the rate of reduction of ferrichrome A, a non-ionophore, was identical with the rate of transport of iron into the cell. Iron(III) citrate was reduced at a rate slightly lower than the rate of transport. These data suggest that reduction of iron(III) is involved in the transport of iron from ferichrome A and possibly from iron(III) citrate.     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria.

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon isotope effects associated with aceticlastic methanogenesis

The carbon isotope effects associated with synthesis of methane from acetate have been determined for Methanosarcina barkeri 227 and for methanogenic archaea in sediments of Wintergreen Lake, Michigan. At 37 degrees C, the 13C isotope effect for the reaction acetate (methyl carbon) --> methane, as measured in replicate experiments with M. barkeri, was - 21.3% +/- 0.3%. The isotope effect at the carboxyl portion of acetate was essentially equal, indicating participation of both positions in the rate-determining step, as expected for reactions catalyzed by carbon monoxide dehydrogenase. A similar isotope effect, - 19.2% +/- 0.3% was found for this reaction in the natural community (temperature = 20 degrees C). Given these observations, it has been possible to model the flow of carbon to methane within lake sediment communities and to account for carbon isotope compositions of evolving methane. Extension of the model allows interpretation of seasonal fluctuations in 13C contents of methane in other systems.     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)