Rate constants for saturation-recovery EPR and ELDOR of 14N-Spin labels.

Saturation-recovery (SR)-EPR can determine electron spin-lattice relaxation rates in liquids over a wide range of effective viscosity, making it especially useful for biophysical and biomedical applications. Here, I develop exact solutions for the SR-EPR and SR-ELDOR rate constants of 14N-nitroxyl spin labels as a function of rotational correlation time and spectrometer operating frequency. Explicit mechanisms for electron spin-lattice relaxation are: rotational modulation of the N-hyperfine and electron-Zeeman anisotropies (specifically including cross terms), spin-rotation interaction, and residual frequency-independent vibrational contributions from Raman processes and local modes. Cross relaxation from mutual electron and nuclear spin flips, and direct nitrogen nuclear spin-lattice relaxation, also must be included. Both the latter are further contributions from rotational modulation of the electron-nuclear dipolar interaction (END). All the conventional liquid-state mechanisms are defined fully by the spin-Hamiltonian parameters; only the vibrational contributions contain fitting parameters. This analysis gives a firm basis for interpreting SR (and inversion recovery) results in terms of additional, less standard mechanisms.

Associations of the Neighborhood Built Environment with Gestational Weight Gain.

OBJECTIVE: This study aimed to determine whether specific factors of the built environment related to physical activity and diet are associated with inadequate and excessive gestational weight gain (GWG). STUDY DESIGN: This analysis is based on data from the Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-To-Be, a prospective cohort of nulliparous women who were followed from the beginning of their pregnancies through delivery. At each study visit, home addresses were recorded and geocoded. Locations were linked to several built-environment characteristics such as the census tract National Walkability Score (the 2010 Walkability Index) and the number of gyms, parks, and grocery stores within a 3-km radius of residential address. The primary outcome of GWG (calculated as the difference between prepregnancy weight and weight at delivery) was categorized as inadequate, appropriate, or excessive based on weight gained per week of gestation. Multinomial regression (generalized logit) models evaluated the relationship between each factor in the built environment and excessive or inadequate GWG. RESULTS: Of the 8,182 women in the analytic sample, 5,819 (71.1%) had excessive GWG, 1,426 (17.4%) had appropriate GWG, and 937 (11.5%) had inadequate GWG. For the majority of variables examined, built environments more conducive to physical activity and healthful food availability were associated with a lower odds of excessive or inadequate GWG category. For example, a higher number of gyms or parks within 3 km of a participant's residential address was associated with lower odds of having excessive (gyms: adjusted odds ratio [aOR] = 0.93 [0.89-0.96], parks: 0.94 [0.90-0.98]) or inadequate GWG (gyms: 0.91 [0.86-0.96]; parks: 0.91 [0.86-0.97]). Similarly, a higher number of grocery stores was associated with lower odds of having excessive GWG (0.94 [0.91-0.97]). CONCLUSION: Among a diverse population of nulliparous women, multiple aspects of the built environment are associated with excessive and inadequate GWG. KEY POINTS: · There are little data on the association between the built environment and pregnancy outcomes.. · Multiple aspects of the built environment are associated with excessive and inadequate GWG.. · These results suggest the role that neighborhood investment may play in improving pregnancy outcomes..

Association of N-Terminal Pro-Brain Natriuretic Peptide Concentration in Early Pregnancy With Development of Hypertensive Disorders of Pregnancy and Future Hypertension.

IMPORTANCE: Hypertensive disorders of pregnancy are associated with future cardiovascular disease, perhaps because of subclinical cardiac dysfunction before pregnancy leading to impaired adaptation to pregnancy. Natriuretic peptides are promising biomarkers for detecting subclinical cardiac dysfunction outside of pregnancy. OBJECTIVE: To investigate whether higher concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) in early pregnancy would be associated with hypertensive disorders of pregnancy and hypertension 2 to 7 years post partum. DESIGN, SETTING, AND PARTICIPANTS: This cohort study used data from the The Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-Be Heart Health Study, a prospective multicenter observational study. A total of 4103 nulliparous women with complete data and no prepregnancy hypertension or diabetes who were treated at 8 clinical sites were included. Women were followed up with for 2 to 7 years after pregnancy. Data were collected from October 2010 to October 2017, and data were analyzed from August 2020 to November 2021. EXPOSURES: NT-proBNP concentration, measured using an electrochemiluminescence immunoassay from a first-trimester blood sample. MAIN OUTCOMES AND MEASURES: Hypertensive disorders of pregnancy and incident hypertension (systolic blood pressure of 130 mm Hg or diastolic blood pressure of 80 mm Hg or use of antihypertensive agents) at follow-up visit. RESULTS: A total of 4103 women met inclusion criteria; the mean (SD) age was 27.0 (5.6) years. Among these women, 909 (22.2%) had an adverse pregnancy outcome, and 817 (19.9%) had hypertension at the follow-up visit. Higher NT-proBNP concentrations were associated with a lower risk of hypertensive disorders of pregnancy (adjusted odds ratio per doubling, 0.81; 95% CI, 0.73-0.91), which persisted after adjustment for age, self-reported race and ethnicity, early-pregnancy body mass index, smoking, and aspirin use. Similarly, higher NT-proBNP concentration in early pregnancy was also associated with a lower risk of incident hypertension 2 to 7 years after delivery (adjusted odds ratio per doubling, 0.84; 95% CI, 0.77-0.93), an association that persisted after controlling for confounders, including hypertensive disorders of pregnancy. CONCLUSIONS AND RELEVANCE: In this cohort study, higher NT-proBNP concentrations in early pregnancy were associated with a lower risk of hypertensive disorders of pregnancy and hypertension 2 to 7 years post partum. These findings suggest that normal early-pregnancy cardiovascular physiology, as assessed by NT-proBNP concentration, may provide biologic insights into both pregnancy outcome and cardiovascular disease risk.

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study.

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.

Associations of the Neighborhood Built Environment With Physical Activity Across Pregnancy.

BACKGROUND: Several features of the neighborhood built environment have been shown to promote leisure-time physical activity (PA) in the general population, but few studies have examined its impact on PA during pregnancy. METHODS: Data were extracted from 8362 Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-Be cohort participants (2010-2013). Residential address information was linked to 3 built environment characteristics: number of gyms and recreation areas within a 3-km radius of residence and census block level walkability. Self-reported leisure-time PA was measured in each trimester and dichotomized as meeting PA guidelines or not. Relative risks for cross-sectional associations between neighborhood characteristics and meeting PA guidelines were estimated using Poisson regression. RESULTS: More gyms and recreation areas were each associated with a greater chance of meeting PA guidelines in models adjusted for sociodemographic characteristics and preexisting conditions. Associations were strongest in the third trimester where each doubling in counts of gyms and recreation areas was associated with 10% (95% confidence interval, 1.07-1.13) and 8% (95% confidence interval, 1.03-1.12), respectively, greater likelihood of meeting PA guidelines. Associations were similar though weaker for walkability. CONCLUSIONS: Results from a large, multisite cohort suggest that these built environment characteristics have similar PA-promoting benefits in pregnant women as seen in more general populations.

Early Pregnancy Atherogenic Profile in a First Pregnancy and Hypertension Risk 2 to 7 Years After Delivery.

Background Cardiovascular risk in young adulthood is an important determinant of lifetime cardiovascular disease risk. Women with adverse pregnancy outcomes (APOs) have increased cardiovascular risk, but the relationship of other factors is unknown. Methods and Results Among 4471 primiparous women, we related first-trimester atherogenic markers to risk of APO (hypertensive disorders of pregnancy, preterm birth, small for gestational age), gestational diabetes mellitus (GDM) and hypertension (130/80 mm Hg or antihypertensive use) 2 to 7 years after delivery. Women with an APO/GDM (n=1102) had more atherogenic characteristics (obesity [34.2 versus 19.5%], higher blood pressure [systolic blood pressure 112.2 versus 108.4, diastolic blood pressure 69.2 versus 66.6 mm Hg], glucose [5.0 versus 4.8 mmol/L], insulin [77.6 versus 60.1 pmol/L], triglycerides [1.4 versus 1.3 mmol/L], and high-sensitivity C-reactive protein [5.6 versus 4.0 nmol/L], and lower high-density lipoprotein cholesterol [1.8 versus 1.9 mmol/L]; P<0.05) than women without an APO/GDM. They were also more likely to develop hypertension after delivery (32.8% versus 18.1%, P<0.05). Accounting for confounders and factors routinely assessed antepartum, higher glucose (relative risk [RR] 1.03 [95% CI, 1.00-1.06] per 0.6 mmol/L), high-sensitivity C-reactive protein (RR, 1.06 [95% CI, 1.02-1.11] per 2-fold higher), and triglycerides (RR, 1.27 [95% CI, 1.14-1.41] per 2-fold higher) were associated with later hypertension. Higher physical activity was protective (RR, 0.93 [95% CI, 0.87-0.99] per 3 h/week). When evaluated as latent profiles, the nonobese group with higher lipids, high-sensitivity C-reactive protein, and insulin values (6.9% of the cohort) had increased risk of an APO/GDM and later hypertension. Among these factors, 7% to 15% of excess RR was related to APO/GDM. Conclusions Individual and combined first-trimester atherogenic characteristics are associated with APO/GDM occurrence and hypertension 2 to 7 years later. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02231398.

Association of Adverse Pregnancy Outcomes With Hypertension 2 to 7 Years Postpartum.

Background Identifying pregnancy-associated risk factors before the development of major cardiovascular disease events could provide opportunities for prevention. The objective of this study was to determine the association between outcomes in first pregnancies and subsequent cardiovascular health. Methods and Results The Nulliparous Pregnancy Outcomes Study Monitoring Mothers-to-be Heart Health Study is a prospective observational cohort that followed 4484 women 2 to 7 years (mean 3.2 years) after their first pregnancy. Adverse pregnancy outcomes (defined as hypertensive disorders of pregnancy, small-for-gestational-age birth, preterm birth, and stillbirth) were identified prospectively in 1017 of the women (22.7%) during this pregnancy. The primary outcome was incident hypertension (HTN). Women without adverse pregnancy outcomes served as controls. Risk ratios (RR) and 95% CIs were adjusted for age, smoking, body mass index, insurance type, and race/ethnicity at enrollment during pregnancy. The overall incidence of HTN was 5.4% (95% CI 4.7% to 6.1%). Women with adverse pregnancy outcomes had higher adjusted risk of HTN at follow-up compared with controls (RR 2.4, 95% CI 1.8-3.1). The association held for individual adverse pregnancy outcomes: any hypertensive disorders of pregnancy (RR 2.7, 95% CI 2.0-3.6), preeclampsia (RR 2.8, 95% CI 2.0-4.0), and preterm birth (RR 2.7, 95% CI 1.9-3.8). Women who had an indicated preterm birth and hypertensive disorders of pregnancy had the highest risk of HTN (RR 4.3, 95% CI 2.7-6.7). Conclusions Several pregnancy complications in the first pregnancy are associated with development of HTN 2 to 7 years later. Preventive care for women should include a detailed pregnancy history to aid in counseling about HTN risk. Clinical Trial Registration URL: http://www.clinicaltrials.gov Unique identifier: NCT02231398.

Autophagy in mammalian neurodevelopment and implications for childhood neurological disorders.

Here we explore the neurodevelopmental aspects of macroautophagy (henceforth known as autophagy), the process by which cells remove and remodel their structure in a regulated and spatially restricted manner. Autophagy is a catabolic pathway in which cytosolic substances, such as protein complexes, lipids, and organelles, are engulfed by an autophagic vesicle. Degradation occurs once an autophagosome fuses with a lysosome, allowing the macromolecular cargo sequestered within the autophagic vesicle to be recycled. It is firmly established that autophagy plays a pivotal role in maintaining cellular homeostasis. Nevertheless, new evidence has emerged that the molecular mechanisms which regulate brain growth and neuronal connectivity involve autophagic processes. Our aim, as we endeavor to review data from model systems, is to show that autophagy performs a fundamental role in the development of the central nervous system (CNS). Moreover, we discuss human genetic data to underscore that mutations in autophagy-related genes are a contributing factor in childhood neurological disorders. To emphasize the importance of regulated vesicle transport pathways during the formation of the CNS, we discuss autophagy in relation to endosomal sorting to the lysosome, and explore how these mechanisms might intersect to regulate developmental events. We maintain that a deeper understanding of the function of autophagy in the CNS can shed new light on the biological basis of neurodevelopmental disorders.

Distinct Populations in Spin-Label EPR Spectra from Nitroxides.

Two-component nitroxide spin-label electron paramagnetic presonance (EPR) spectra are important to the analysis of lipid-protein interactions, phase separation in lipid membranes, and conformational changes in proteins. A paper published in this journal offers an interpretation of such spectra based on simulations with single-site models. It is not possible to reproduce those simulations published in Meirovitch , E. ; [ Analysis of Protein Lipid Interactions Based on Model Simulations of Electron Spin Resonance Spectra . J. Phys. Chem. 1984 , 88 , 3454 - 3465 ] that might conceivably be taken to resemble two-component line shapes, when using the motional model and parameters given in that paper. Instead of the apparent two components, the spectra resemble single-component powder patterns expected from axially anisotropic, partial motional-averaging (a situation familiar for chain-labeled lipids in nonaligned fluid membranes). This is because: (i) the nitroxide z-axis is inclined at a fixed angle to the principal diffusion axis, and (ii) motion perpendicular to the principal diffusion axis is so slow as to approximate a powder distribution. The line shapes are compatible with simulations that use the same model for a complete range of nitroxide order parameters [ Schorn , K. ; Extracting Order Parameters from Powder EPR Lineshapes for Spin-Labelled Lipids in Membranes . Spectrochim. Acta, Part A 1997 , 53 , 2235 - 2240 ], which are able to describe single-component experimental spectra from lipids spin-labeled at different chain positions in fluid-bilayer membranes [ Schorn , K. ; Lipid Chain Dynamics in Diacylglycerol-Phosphatidylcholine Mixtures Studied by Slow-Motional Simulations of Spin Label ESR Spectra . Chem. Phys. Lipids 1996 , 82 , 7 - 14 ]. Neither from simulation nor from experiment is there any basis to assert that single-component nitroxyl EPR spectra resemble those containing two components.

Prescription and Other Medication Use in Pregnancy.

OBJECTIVE: To characterize prescription and other medication use in a geographically and ethnically diverse cohort of women in their first pregnancy. METHODS: In a prospective, longitudinal cohort study of nulliparous women followed through pregnancy from the first trimester, medication use was chronicled longitudinally throughout pregnancy. Structured questions and aids were used to capture all medications taken as well as reasons they were taken. Total counts of all medications taken including number in each category and class were captured. Additionally, reasons the medications were taken were recorded. Trends in medications taken across pregnancy and in the first trimester were determined. RESULTS: Of the 9,546 study participants, 9,272 (97.1%) women took at least one medication during pregnancy with 9,139 (95.7%) taking a medication in the first trimester. Polypharmacy, defined as taking at least five medications, occurred in 2,915 (30.5%) women. Excluding vitamins, supplements, and vaccines, 73.4% of women took a medication during pregnancy with 55.1% taking one in the first trimester. The categories of drugs taken in pregnancy and in the first trimester include the following: gastrointestinal or antiemetic agents (34.3%, 19.5%), antibiotics (25.5%, 12.6%), and analgesics (23.7%, 15.6%, which includes 3.6%; 1.4% taking an opioid pain medication). CONCLUSION: In this geographically and ethnically diverse cohort of nulliparous pregnant women, medication use was nearly universal and polypharmacy was common. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT01322529.

Lipid Configurations from Molecular Dynamics Simulations.

The extent to which current force fields faithfully reproduce conformational properties of lipids in bilayer membranes, and whether these reflect the structural principles established for phospholipids in bilayer crystals, are central to biomembrane simulations. We determine the distribution of dihedral angles in palmitoyl-oleoyl phosphatidylcholine from molecular dynamics simulations of hydrated fluid bilayer membranes. We compare results from the widely used lipid force field of Berger et al. with those from the most recent C36 release of the CHARMM force field for lipids. Only the CHARMM force field produces the chain inequivalence with sn-1 as leading chain that is characteristic of glycerolipid packing in fluid bilayers. The exposure and high partial charge of the backbone carbonyls in Berger lipids leads to artifactual binding of Na+ ions reported in the literature. Both force fields predict coupled, near-symmetrical distributions of headgroup dihedral angles, which is compatible with models of interconverting mirror-image conformations used originally to interpret NMR order parameters. The Berger force field produces rotamer populations that correspond to the headgroup conformation found in a phosphatidylcholine lipid bilayer crystal, whereas CHARMM36 rotamer populations are closer to the more relaxed crystal conformations of phosphatidylethanolamine and glycerophosphocholine. CHARMM36 alone predicts the correct relative signs of the time-average headgroup order parameters, and reasonably reproduces the full range of NMR data from the phosphate diester to the choline methyls. There is strong motivation to seek further experimental criteria for verifying predicted conformational distributions in the choline headgroup, including the 31P chemical shift anisotropy and 14N and CD3 NMR quadrupole splittings.

Molecular order and T1-relaxation, cross-relaxation in nitroxide spin labels.

Interpretation of saturation-recovery EPR experiments on nitroxide spin labels whose angular rotation is restricted by the orienting potential of the environment (e.g., membranes) currently concentrates on the influence of rotational rates and not of molecular order. Here, I consider the dependence on molecular ordering of contributions to the rates of electron spin-lattice relaxation and cross relaxation from modulation of N-hyperfine and Zeeman anisotropies. These are determined by the averages 〈cos2θ〉 and 〈cos4θ〉, where θ is the angle between the nitroxide z-axis and the static magnetic field, which in turn depends on the angles that these two directions make with the director of uniaxial ordering. For saturation-recovery EPR at 9 GHz, the recovery rate constant is predicted to decrease with increasing order for the magnetic field oriented parallel to the director, and to increase slightly for the perpendicular field orientation. The latter situation corresponds to the usual experimental protocol and is consistent with the dependence on chain-labelling position in lipid bilayer membranes. An altered dependence on order parameter is predicted for saturation-recovery EPR at high field (94 GHz) that is not entirely consistent with observation. Comparisons with experiment are complicated by contributions from slow-motional components, and an unexplained background recovery rate that most probably is independent of order parameter. In general, this analysis supports the interpretation that recovery rates are determined principally by rotational diffusion rates, but experiments at other spectral positions/field orientations could increase the sensitivity to order parameter.

Ether-linked lipids: Spin-label EPR and spin echoes.

Electron spin echo envelope modulation (ESEEM) and conventional electron paramagnetic resonance (EPR) of site-specifically spin-labelled phospholipids are used to investigate the effect of ether-linked chains on the water-penetration and polarity profiles, as well as the phase behaviour and chain flexibility profiles, of phospholipid membranes. D2O-ESEEM reveals that water exposure of the terminal methyl groups in the interdigitated phase of dihexadecyl phosphatidylcholine (DHPC) is comparable to that of the methylene groups at the polar head-group end of the chains. Similarly, an uniform transmembrane polarity profile is obtained from the dependence of the outer 14N-hyperfine splitting on the spin-label position along the chain in frozen interdigitated DHPC dispersions. Two-component conventional EPR spectra of spin labels at the terminal methyl end of the chain reveal that the intermediate gel phase above the pretransition of DHPC contains components in which the lipid chains are interdigitated. The polarity and chain-flexibility profiles in the fluid Lα-phase of DHPC with ether-linked chains are shifted outwards, towards the polar-apolar interface, as compared with that of dihexadecanoyl phosphatidylcholine (DPPC) with ester-linked chains. Also, the polarity profile of DHPC is shifted upwards, to higher polarities. These differences reflect those in hydrocarbon thickness and area/lipid molecule reported by x-ray diffraction for the Lα-phases of the two lipids.

Spin-label Order Parameter Calibrations for Slow Motion.

Calibrations are given to extract orientation order parameters from pseudo-powder electron paramagnetic resonance line shapes of 14N-nitroxide spin labels undergoing slow rotational diffusion. The nitroxide z-axis is assumed parallel to the long molecular axis. Stochastic-Liouville simulations of slow-motion 9.4-GHz spectra for molecular ordering with a Maier-Saupe orientation potential reveal a linear dependence of the splittings, [Formula: see text] and [Formula: see text], of the outer and inner peaks on order parameter [Formula: see text] that depends on the diffusion coefficient [Formula: see text] which characterizes fluctuations of the long molecular axis. This results in empirical expressions for order parameter and isotropic hyperfine coupling: [Formula: see text] and [Formula: see text], respectively. Values of the calibration constants [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text] are given for different values of [Formula: see text] in fast and slow motional regimes. The calibrations are relatively insensitive to anisotropy of rotational diffusion [Formula: see text], and corrections are less significant for the isotropic hyperfine coupling than for the order parameter.

Coherence transfer and electron T1-, T2-relaxation in nitroxide spin labels.

Abragam's double-commutator spin operator method is used to analyse: 1) electron coherence transfer by intermolecular dipolar interaction between spin-label radicals, and 2) longitudinal and transverse electron spin relaxation by rotational modulation of the Zeeman and nitrogen-hyperfine anisotropies of isolated nitroxide spin labels. Results compatible with earlier treatments by Redfield theory are obtained without specifically evaluating matrix elements. Extension to single-transition operators for isolated nitroxides predicts electron coherence transfer by pseudosecular electron-nuclear dipolar interaction, in the absence of intermolecular dipolar coupling. This explains earlier experimental findings that coherence transfer (specifically dispersion-like distortion of the EPR absorption line shape) does not extrapolate to zero at low concentrations of nitroxide spin labels.

Nuclear spin-lattice relaxation in nitroxide spin-label EPR.

Nuclear relaxation is a sensitive monitor of rotational dynamics in spin-label EPR. It also contributes competing saturation transfer pathways in T1-exchange spectroscopy, and the determination of paramagnetic relaxation enhancement in site-directed spin labelling. A survey shows that the definition of nitrogen nuclear relaxation rate Wn commonly used in the CW-EPR literature for 14N-nitroxyl spin labels is inconsistent with that currently adopted in time-resolved EPR measurements of saturation recovery. Redefinition of the normalised 14N spin-lattice relaxation rate, b=Wn/(2We), preserves the expressions used for CW-EPR, whilst rendering them consistent with expressions for saturation recovery rates in pulsed EPR. Furthermore, values routinely quoted for nuclear relaxation times that are deduced from EPR spectral diffusion rates in 14N-nitroxyl spin labels do not accord with conventional analysis of spin-lattice relaxation in this three-level system. Expressions for CW-saturation EPR with the revised definitions are summarised. Data on nitrogen nuclear spin-lattice relaxation times are compiled according to the three-level scheme for 14N-relaxation: T1n=1/Wn. Results are compared and contrasted with those for the two-level 15N-nitroxide system.

Cross relaxation in nitroxide spin labels.

Cross relaxation, and mI-dependence of the intrinsic electron spin-lattice relaxation rate We, are incorporated explicitly into the rate equations for the electron-spin population differences that govern the saturation behaviour of 14N- and 15N-nitroxide spin labels. Both prove important in spin-label EPR and ELDOR, particularly for saturation recovery studies. Neither for saturation recovery, nor for CW-saturation EPR and CW-ELDOR, can cross relaxation be described simply by increasing the value of We, the intrinsic spin-lattice relaxation rate. Independence of the saturation recovery rates from the hyperfine line pumped or observed follows directly from solution of the rate equations including cross relaxation, even when the intrinsic spin-lattice relaxation rate We is mI-dependent.