Structure of melittin bound to phospholipid micelles studied using hydrogen-deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The structure of melittin bound to dodecylphosphocholine (DPC) micelles was investigated using hydrogen–deuterium (H/D) exchange in conjunction with collision induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of melittin with or without DPC micelles was analyzed at different time points examining the mass of each fragment ion produced by hexapole CID. When melittin existed alone in aqueous solution, more than 80% of amide hydrogens was exchanged within 10 s, and the deuterium content in each fragment ion showed high values throughout the experiments. When melittin was bound to DPC micelles, the percentage of deuterium incorporation into the fragment decreased remarkably at any time point. It increased little by little as the exchange period prolonged, indicating that some stable structure was formed by the interaction with DPC. The results obtained here were consistent with the previous studies on the helical structure of melittin carried out by NMR and CD analyses. The strategy using H/D exchange and MS analysis might be useful for studying structural changes of peptides and proteins caused by phospholipid micelles. It could also be applied to membrane-bound proteins to characterize their structure.     

Improving hydrogen/deuterium exchange mass spectrometry by reduction of the back-exchange effect

The measurement of deuterium incorporation kinetics using hydrogen/deuterium (H/D) exchange experiments is a valuable tool for the investigation of the conformational dynamics of biomolecules in solution. Experiments consist of two parts when using H/D exchange mass spectrometry to analyse the deuterium incorporation. After deuterium incorporation at high D(2)O concentration, it is necessary to decrease the D(2)O concentration before the mass analysis to avoid deuterium incorporation under artificial conditions of mass spectrometric preparation and measurement. A low D(2)O concentration, however, leads to back-exchange of incorporated deuterons during mass analysis. This back-exchange is one of the major problems in H/D exchange mass spectrometry and must be reduced as much as possible. In the past, techniques using electrospray ionization (ESI) had the lowest back-exchange values possible in H/D exchange mass spectrometry. Methods for the measurement of H/D exchange by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that have been developed since 1998 have some significant advantages, but they could not achieve the back-exchange minima of ESI methods. Here, we present a protocol for H/D exchange MALDI-MS which allows for greater minimization of back-exchange compared with H/D exchange ESI-MS under similar conditions.     

Studies on the biosynthesis of phomoidride B (CP-263,114): evidence for a decarboxylative homodimerization pathway

[reaction: see text]. Feeding experiments using a deuterium labeled C16 maleic anhydride and whole cell culture of ATCC 74256 led to the isolation of phomoidride B with deuterium incorporation at C(7) and C(19) as determined by 2H NMR and electrospray mass spectrometry. This result is in accord with a decarboxylative homodimerization of the C16 maleic anhydride as a key biosynthetic step leading to phomoidride B.     

Total synthesis of 2',3',4',5',5' '-(2)h(5)-ribonucleosides: the key building blocks for NMR structure elucidation of large RNA

The diastereospecific chemical syntheses of uridine-2',3',4',5',5' '-(2)H(5) (21a), adenosine-2',3',4',5',5' '-(2)H(5) (21b), cytidine-2',3',4',5',5' '-(2)H(5)(2)H(5) (21c), and guanosine-2',3',4',5',5' '-(2)H(5) (21d) (>97 atom % (2)H at C2', C3', C4', and C5'/C5' ') have been achieved for their use in the solution NMR structure determination of oligo-RNA by the Uppsala "NMR-window" concept (refs 4a-c, 5a, 6), in which a small (1)H segment is NMR-visible, while the rest is made NMR-invisible by incorporation of the deuterated blocks 21a-d. The deuterated ribonucleosides 21a-d have been prepared by the condensation of appropriately protected aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-alpha/beta-D-ribofuranose-2,3,4,5,5'-(2)H(5) (19), which has been obtained via diastereospecific deuterium incorporation at the C2 center of appropriate D-ribose-(2)H(4) derivatives either through an oxidation-reduction-inversion sequence or a one-step deuterium-proton exchange in high overall yield (44% and 24%, respectively).     

Site-specific amide hydrogen/deuterium exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry

Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E. coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels. Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.     

液体样品对核磁图谱质量影响因素探讨

结合样品核磁测试结果,以具体实例的形式,总结了核磁样品分析的基本要求,分析了核磁管和氘代试剂的选择、样品制备及pH值等因素对核磁测试结果的影响,并提出了样品最优测试条件:核磁管管体内外表面要足够光洁均匀,在高温条件下,优先选用升温核磁管.尽量选择小支封装氘代试剂,降低水峰的影响,并且应考虑样品的性质,避免试剂与测试样品发生反应.样品必须与溶剂充分混合均匀,样品量要在溶液粘度与灵敏度之间做好平衡.在分析与氧、氮和硫共价相连的氢原子时,应该选择恰当的pH溶液.以上研究结果可以为其它样品核磁测试提供借鉴指导.     

New insights on the mechanism of palladium-catalyzed hydrolysis of sodium borohydride from 11B NMR measurements

To gain insight on the mechanistic aspects of the palladium-catalyzed hydrolysis of NaBH(4) in alkaline media, the kinetics of the reaction has been investigated by (11)B NMR (nuclear magnetic resonance) measurements taken at different times during the reaction course. Working with BH(4)(-) concentration in the range 0.05-0.1 M and with a [substrate]/[catalyst] molar ratio of 0.03-0.11, hydrolysis has been found to follow a first-order kinetic dependence from concentration of both the substrate and the catalyst (Pd/C 10 wt %). We followed the reaction of NaBH(4) and its perdeuterated analogue NaBD(4) in H(2)O, in D(2)O and H(2)O/D(2)O mixtures. When the process was carried out in D(2)O, deuterium incorporation in BH(4)(-) afforded BH(4)(-)(n)D(n)(-) (n = 1, 2, 3, 4) species, and a competition between hydrolysis and hydrogen/deuterium exchange processes was observed. By fitting the kinetics NMR data by nonlinear least-squares regression techniques, the rate constants of the elementary steps involved in the palladium-catalyzed borohydride hydrolysis have been evaluated. Such a regression analysis was performed on a reaction scheme wherein the starting reactant BH(4)(-) is allowed both to reversibly exchange hydrogen with deuterium atoms of D(2)O and to irreversibly hydrolyze into borohydroxy species B(OD)(4)(-). In contrast to acid-catalyzed hydrolysis of sodium borohydride, our results indicate that in the palladium-catalyzed process the rate constants of the exchange processes are higher than those of the corresponding hydrolysis reactions.     

Fourier transform mass spectrometry to monitor hyaluronan-protein interactions: use of hydrogen/deuterium amide exchange

The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.     

High-resolution H/D exchange studies on the HET-s218-295 prion protein

In a search for improved resolution of hydrogen/deuterium (H/D) exchange experiments analyzed by mass spectrometry (HXMS), we evaluated two methodologies for a detailed structural study of solvent accessibility in the case of the HET-s(218-295) prion protein. For the first approach, after incubation in the deuterated solvent, aggregated HET-s(218-295) was digested with pepsin and the generated peptides were analyzed by nanospray mass spectrometry in an ion trap, with and without collision-induced dissociation (CID). We compared deuterium incorporation in peptides as determined on peptide pseudomolecular ions and on b and y fragments produced by longer peptides under CID conditions. For both b and y fragment ions, an extensive H/D scrambling phenomenon was observed, in contrast with previous studies comparing CID-MS experiments and (1)H NMR data. Thus, the spatial resolution of HXMS experiments could not be improved by means of MS/MS data generated by an ion trap mass spectrometer. In a second approach, the incorporation of deuterium was analyzed by MS for 76 peptides of the HET-s(218-289) peptide mass fingerprint, and the use of shared boundaries among peptic peptides allowed us to determine deuteration levels of small regions ranging from one to four amino acids. This methodology led to evidence of highly protected regions along the HET-s(218-295) sequence.     

Characterization of a chiral enolate aggregate and observation of 6Li-1H scalar coupling

A chiral enolate aggregate 1 containing a lithium enolate and a chiral lithium amide was systematically investigated by various NMR techniques. (1)H and (13)C DOSY at 25 and -78 degrees C provide its solution structure, aggregation number, and formula weight. Multiple 2D (6)Li NMR techniques, such as (6)Li-(6)Li EXSY, (6)Li-(1)H HOESY, were utilized to investigate its stereochemical structure. The configuration of the enolate in complex 1 was confirmed by (6)Li-(1)H HOESY and trapping with TMS-Cl. A unique (6)Li-(1)H coupling through the Li-N-C-H network was observed. This scalar coupling was corroborated by (6)Li-(1)H HMQC, deuterium labeling experiments, and selective (1)H decoupling (6)Li NMR. The stereostructure of 1 provides a model for the origin of enantioselectivity of chiral lithium amide-induced enolate addition reactions.     

含活泼质子水溶性化合物的核磁共振结构解析

利用核磁共振对在水溶剂易溶且含活泼质子的化合物的结构进行解析.实验中将待测样品直接溶解于二次去离子水中,并利用外标进行锁场.这样既保证了核磁共振实验的正常进行,同时避免了实验中氘代试剂对活泼质子的置换效应,为化合物中活泼质子的定位提供了直接的依据.为了保证外标锁场实验核磁共振谱图的质量,本工作采用了溶剂峰压制的核磁共振实验技术,获取了一组高质量的1D、2D谱图,并在此基础上顺利完成了对头孢米诺盐的结构确定.     

Ring strain and its effect on the rate of the general-base catalyzed enolization of cyclobutanone

[reaction: see text] The 3-quinuclidinone-catalyzed (pK(BH) = 7.5) enolization of cyclobutanone (1) in D(2)O at 25 degrees C, I = 1.0 (KCl) was followed by deuterium incorporation, which was determined by (1)H NMR. The second-order rate constant for the buffer-catalyzed deprotonation of 1 was found to be k(B) = 3.3 x 10(-4) M(-1) s(-1), which is compared to rates for acetone and 2-(2'-oxopropyl)benzaldehyde under similar conditions. The data shows that ring strain has very little effect on the energy barrier to deprotonation of 1 vs the unstrained systems.     

Molecular hydrogen tweezers: structure and mechanisms by neutron diffraction, NMR, and deuterium labeling studies in solid and solution

The mechanism of reversible hydrogen activation by ansa-aminoboranes, 1-N-TMPH-CH(2)-2-[HB(C(6)F(5))(2)]C(6)H(4) (NHHB), was studied by neutron diffraction and thermogravimetric mass-spectroscopic experiments in the solid state as well as with NMR and FT-IR spectroscopy in solution. The structure of the ansa-ammonium borate NHHB was determined by neutron scattering, revealing a short N-H···H-B dihydrogen bond of 1.67 ?. Moreover, this intramolecular H-H distance was determined in solution to be also 1.6-1.8 ? by (1)H NMR spectroscopic T(1) relaxation and 1D NOE measurements. The X-ray B-H and N-H distances deviated from the neutron and the calculated values. The dynamic nature of the molecular tweezers in solution was additionally studied by multinuclear and variable-temperature NMR spectroscopy. We synthesized stable, individual isotopic isomers NDDB, NHDB, and NDHB. NMR measurements revealed a primary isotope effect in the chemical shift difference (p)Δ(1)H(D) = δ(NH) - δ(ND) (0.56 ppm), and hence supported dihydrogen bonding. The NMR studies gave strong evidence that the structure of NHHB in solution is similar to that in the solid state. This is corroborated by IR studies providing clear evidence for the dynamic nature of the intramolecular dihydrogen bonding at room temperature. Interestingly, no kinetic isotope effect was detected for the activation of deuterium hydride by the ansa-aminoborane NB. Theoretical calculations attribute this to an "early transition state". Moreover, 2D NOESY NMR measurements support fast intermolecular proton exchange in aprotic CD(2)Cl(2) and C(6)D(6).     

Fragmentation of the lamellae and fractionation of polymer coils upon mixing poly(dimethylacrylamide) with the lamellar phase of aerosol OT in water

The lamellar mesophase formed by surfactant 1,4-bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in deuterated water is mixed with poly(dimethylacrylamide) (PDMAA) polymers of low molecular weight (Mn= (2-20) x 10(3)). The mixtures separate into microphases (lamellar plus isotropic polymer solution). Their microstructures are studied by microscopy, small-angle X-ray scattering (SAXS), and deuterium NMR (2H NMR). According to SAXS, the lamellar phase fractionates the molecular weight distribution of the polymer, by dissolving only chains with coil sizes smaller than the thickness of the water layers between lamellae, and keeping larger chains segregated from the lamellar phase. The fraction of polymer that is segregated from the lamellar phase grows with Mn of the polymer. In 2H NMR, there are two signals, a quadrupolar doublet (water molecules hydrating the anisotropic lamellar phase contribute to this doublet) and a singlet (water molecules in the isotropic polymer solution contribute to this singlet). These two signals are deconvoluted to analyze the phases. Mixing with the polymer produces the partial dispersion of the lamellar phase into small fragments (microcrystallites). The structure of these microcrystallites is such that they conserve the regular long period spacing of the macrophase, and are thus identified in SAXS, but they are smaller than the minimum size required to produce quadrupolar splitting (about 4 microm), and therefore, in 2H NMR, they contribute to the singlet. 2H NMR can thus not distinguish between small microcrystallites and an isotropic polymer solution segregated from the lamellar phase; instead small microcrystallites are detected as an apparent increase of the isotropic solution. The degree of dispersion produced by the polymer in the lamellar phase is correlated with the degree of segregation that the polymer suffers. Thus, much greater dispersion into microcrystallites is produced by the higher Mn polymers than by the lower Mn polymers (in the range covered by the present samples, although with a much higher molecular weight sample (3 x 10(6)) that is totally segregated no such microcrystallites were detected).     

Deuterium attachment to carbon nanotubes in deuterated water

A systematic investigation on the unusual attachment of labile deuterium to carbon nanotubes in deuterated water and alcohols is reported. The carbon nanotubes were solubilized through the established functionalization of the nanotube-bound carboxylic acids to allow solution-phase reaction and characterization. The deuterium attachment was found under several experimental conditions, including the use of deuterated ethanol as a co-reactant in the nanotube functionalization reaction and the refluxing of functionalized or simply purified carbon nanotubes in deuterated water and alcohols. The solubility of the functionalized carbon nanotube samples in common organic solvents and water allowed unambiguous (2)H NMR characterization. The reproducible broad (2)H NMR signal at approximately 6.5 ppm is assigned to carbon nanotube-attached deuterium species. The assignment is supported by the results from FT-IR measurements. The carbon-deuterium interaction is so strong that the corresponding vibration resembles the typical C-D stretching mode in the characteristic frequency region. The FT-IR peak intensities also correlate well with the (2)H NMR signal integrations in a series of samples. Mechanistic implications of the results are discussed.     

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of human cardiac troponin C

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.     

Dynamics of dilute water in carbon tetrachloride

A dilute solution of water in a hydrophobic solvent, such as carbon tetrachloride (CCl4), presents an opportunity to study the rotational properties of water without the complicating effects of hydrogen bonds. We report here the results of theoretical, experimental, and semiempirical studies of a 0.03 mole percent solution of water in CCl4. It is shown that for this solution there are negligible water-water interactions or water-CCl4 interactions; theoretical and experimental values for proton NMR chemical shifts (deltaH) are used to confirm the minimal interactions between water and the CCl4. Calculated ab initio values and semiempirical values for oxygen-17 and deuterium quadrupole coupling constants (chi) of water/CCl4 clusters are reported. Experimental values for the 17O, 2H, and 1H NMR spin-lattice relaxation times, T1, of 0.03 mole percent water in dilute CCl4 solution at 291 K are 94+/-3 ms, 7.0+/-0.2 s, and 12.6+/-0.4 s, respectively. These T1 values for bulk water are also referenced. "Experimental" values for the quadrupole coupling constants and relaxation times are used to obtain accurate, experimental values for the rotational correlation times for two orthogonal vectors in the water molecule. The average correlation time, tauc, for the position vector of 17O (orthogonal to the plane of the molecule) in monomer water, H2(17)O, is 91 fs. The average value for the deuterium correlation time for the deuterium vector in 2H2O is 104 fs; this vector is along the OD bond. These values indicate that the motion of monomer water in CCl4 is anisotropic. At 291 K, the oxygen rotational correlation time in bulk 2H2(17)O is 2.4 ps, the deuterium rotational correlation time in the same molecule is 3.25 ps. (Ropp, J.; Lawrence, C.; Farrar, T. C.; Skinner, J. L. J. Am. Chem. Soc. 2001, 123, 8047.) These values are a factor of about 20 longer than the tauc value for dilute monomer water in CCl4.     

Investigations of polypeptide rotational diffusion in aligned membranes by 2H and 15N solid-state NMR spectroscopy

Transmembrane and in-plane oriented peptides have been prepared by solid-phase peptide synthesis, labeled with 3,3,3-2H3-alanine and 15N-leucine at two selected sites, and reconstituted into oriented phophatidylcholine membranes. Thereafter, proton-decoupled 15N and 2H solid-state NMR spectroscopy at sample orientations of the membrane normal parallel to the magnetic field direction have been used to characterize the tilt and rotational pitch angle of these peptides in some detail. In a second step the samples have been tilted by 90 degrees . In this setup the spectral line shapes are sensitive indicators of the rate of rotational diffusion. Whereas monomeric transmembrane peptides exhibit spectral averaging and well-defined resonances, larger complexes are characterized by broad spectral line shapes. In particular the deuterium line shape is sensitive to association of a few transmembrane helices. In contrast, the formation of much larger complexes affects the 15N chemical shift spectrum. The spectra indicate that in liquid crystalline membranes an amphipathic peptide of 14 amino acids exhibits fast rotational diffusion on both the 2H and 15N time scales (>10(-5) s). Extending the sequences to 26 amino acids results in pronounced changes of the 2H solid-state NMR spectrum, whereas the signal intensities of 15N solid-state NMR spectra degrade. Below the phase transition temperature of the phospholipid bilayers, motional averaging on the time scale of the 2H solid-state NMR spectrum ceases for transmembrane and in-plane oriented peptides. Furthermore at temperatures close to the phase transition the total signal intensities of the deuterium solid-state NMR spectra strongly decrease.     

trans-W(Cmesityl)(dmpe)2H: revealing a highly polar W-H bond and H-mobility in liquid and solid state

The isotopomeric complexes trans-W(Cmesityl)[(C(H,D)3)2PCH2CH2P(C(H,D)3)2]2(H,D) 1-4 were prepared. 2 (W(Cmesityl)(dmpe)2D) was used to study the Deuterium Quadrupole Coupling Constant (DQCC) and the ionicity of the W-D bond (DQCC=34.1 kHz; ionicity 85%). 1 (W(Cmesityl)(dmpe)2H) shows several dynamic exchange processes in solution, such as HW/HW, HW/ortho-Memesityl, and HW/H2 exchanges observed by NMR in combination with deuterium labeling studies and double label crossover experiments. Except for the HW/H2, these reactions comprise elementary steps, which also appear along the isomerization pathway of 1 into (2,3,5-trimethylphenylcarbyne)(dmpe)2WH (5) at 60 degrees C. 5 was characterized by an X-ray diffraction study. In the solid state only an HW/Mep exchange process prevails appearing at higher temperatures, which was identified by NMR and by Quasielastic Neutron Scattering. The latter also provided an activation barrier of 5 kcal/mol and a "jump width" for the moving H nucleus in agreement with the HW...Mep distance of the X-ray diffraction study of 1.     

Measurement of pulmonary surfactant disaturated-phosphatidylcholine synthesis in human infants using deuterium incorporation from body water

The aim of the study was to determine surfactant palmitate disaturated-phosphatidylcholine (DSPC-PA) synthesis in vivo in humans by the incorporation of deuterium from total body water into DSPC-PA under steady state condition. We studied three newborns and one infant (body weight (BW) 4.6 +/- 2.9 kg, gestational age 37.5 +/- 2 weeks, age 9 +/- 9 days) and four preterm newborns (BW 1.3 +/- 0.6 kg, gestational age 30.3 +/- 2.5 weeks, postnatal age 8.8 +/- 9.2 h). All infants were mechanically ventilated during the study and the four preterm infants received exogenous surfactant at the start of the study.We administered 0.44 g (2)H(2)O/kg BW as a bolus intravenously, followed by 0.0125 g (2)H(2)O/kg BW every 6 h to maintain deuterium enrichment at plateau over 2 days. Urine samples and tracheal aspirates (TA) were obtained prior to dosing and every 6 h thereafter. Isotopic enrichment curves of DSPC-PA from sequential TA and urine deuterium enrichments were analyzed by Gas Chromatography-Isotope Ratio-Mass Spectrometry (GC-IRMS) and normalized for Vienna Standard Mean Ocean Water. Enrichment data were used to measure DSPC-PA fractional synthesis rate (FSR) from the linear portion of the DSPC-PA enrichment rise over time, relative to plateau enrichment of urine deuterium. Secretion time (ST) was defined as the time lag between the start of the study and the appearance of DSPC-PA deuterium enrichment in TA. Data were given as mean +/- SD.All study infants reached deuterium-steady state in urine. DSPC-PA FSR was 6.5 +/- 2.8%/day (range 2.6-10.2). FSR for infants who did not receive exogenous surfactant was 5.7 +/- 3.5%/day (range 2.6-9.9%/day) and 7.3 +/- 2.1%/day (range 5.1-10.2%/day) in the preterms, whereas DSPC-PA ST was 10 +/- 10 h and 31 +/- 10 h respectively.Surfactant DSPC-PA synthesis can be measured in humans by the incorporation of deuterium from body water. This study is a simpler and less invasive method compared to previously published methods on surfactant kinetics by means of stable isotopes.