Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.     

New insight into phase behavior and permeability of skin lipid models based on sphingosine and phytosphingosine ceramides

The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment.     

Infrared spectroscopy studies of mixtures prepared with synthetic ceramides varying in head group architecture: Coexistence of liquid and crystalline phases

The barrier function of the skin is provided by the stratum corneum (SC), the outermost layer of the skin. Ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) are present in SC and form highly ordered crystalline lipid lamellae. These lamellae are crucial for a proper skin barrier function. In the present study, Fourier transform infrared spectroscopy was used to examine the lipid organization of mixtures prepared from synthetic CERs with CHOL and FFAs. The conformational ordering and lateral packing of these mixtures showed great similarities to the lipid organization in SC and lipid mixtures prepared with native CERs. Therefore, mixtures with synthetic CERs serve as an excellent tool for studying the effect of molecular architecture of CER subclasses on the lipid phase behavior. In SC the number of OH-groups in the head groups of CER subclasses varies. Furthermore, acylCERs with a linoleic acid chemically bound to a long acyl chain are also identified. The present study revealed that CER head group architecture affects the lateral packing and conformational ordering of the CER:CHOL:FFA mixtures. Furthermore, while the majority of the lipids form a crystalline packing, the linoleate moiety of the acylCERs participates in a “pseudo fluid” phase.     

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

Impact of the long chain ω-acylceramides on the stratum corneum lipid nanostructure. Part 1: Thermotropic phase behaviour of CER[EOS] and CER[EOP] studied using X-ray powder diffraction and FT-Raman spectroscopy

The stratum corneum (SC), the outermost layer of the mammalian skin, is the main skin barrier. Ceramides (CERs) as the major constituent of the SC lipid matrix are of particular interest. At the moment, 11 classes of CERs are identified, but the effect of each single ceramide species is still not known.Therefore in this article, the thermotropic behaviour of the long chain ω-acylceramides CER[EOS] and CER[EOP] was studied using X-ray powder diffraction and FT-Raman spectroscopy.It was found that the ω-acylceramides CER[EOS] and CER[EOP] do not show a pronounced polymorphism which is observed for shorter chain ceramides as a significant feature. The phase behaviour of both ceramides is strongly influenced by the extremely long acyl-chain residue. The latter has a much stronger influence compared with the structure of the polar head group, which is discussed as extremely important for the appearance of a rich polymorphism. Despite the strong influence of the long chain, the additional OH-group of the phyto-sphingosine type CER[EOP] influences the lamellar repeat distance and the chain packing. The less polar sphingosine type CER[EOS] is stronger influenced by the long acyl-chain residue. Hydration is necessary for the formation of an extended hydrogen-bonding network between the polar head groups leading to the appearance of a long-periodicity phase (LPP). In contrast, the more polar CER[EOP] forms the LPP with densely packed alkyl chains already in the dry state.     

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.     

Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair

Ceramides (CERs) play key roles in signal transduction and cell regulation, probably during the keratinization of human hair. Current methods using mass spectrometry (MS), however, are not sufficient to allow the comprehensive analysis of CER molecules, including isobaric and isomeric CERs. Therefore, a method for the comprehensive profiling of CERs was developed. The method developed is based on reversed-phase liquid chromatography (RPLC) coupled to atmospheric pressure chemical ionization (APCI)-MS. Comprehensive identification and profiling of CERs is achieved using two sets of multimass chromatograms obtained from two channel detections that monitor both molecular-related and sphingoid-related ions under two different in-source collision-induced dissociation conditions and using retention times obtained from RPLC. The application of this method revealed that human hair contains 73 species of CER molecules, which were all corroborated by structural analysis using tandem mass spectrometry. The results further revealed that the composition is characterized by predominant molecules consisting of even carbon atom-containing saturated/unsaturated nonhydroxy or alpha-hydroxy fatty acids and C(18) dihydrosphingosine, a minor but distinct content of isobaric/isomeric and odd chain-containing CERs. This successfully developed RPLC-APCI-MS technique allows the comprehensive profiling of CER molecules in hair for the investigation of their physicochemical and physiological roles.     

An enhanced plant lipidomics method based on multiplexed liquid chromatography–mass spectrometry reveals additional insights into cold‐ and drought‐induced membrane remodeling

Within the lipidome of plants a few bulk molecular species hamper the detection of the rest, which are present at relatively low levels. In addition, low‐abundance species are often masked by numerous isobaric interferences, such as those caused by isoelemental species and isotopologues. This scenario not only means that minor species are underrepresented, but also leads to potential misidentifications and limits the structural information gathered by lipidomics approaches. In order to overcome these limitations we have developed a multiplexed liquid chromatography–mass spectrometry lipidomics platform able to achieve an enhanced coverage of plant lipidomes. The platform is based on a single extraction step followed by a series of ultra‐performance liquid chromatography separations. Post‐column flow is then directed to both a triple quadrupole analyzer for targeted profiling and a time‐of‐flight analyzer for accurate mass analysis. As a proof of concept, plants were subjected to cold or drought, which are known to trigger widespread remodeling events in plant cell membranes. Analysis of the leaf lipidome yielded 393 molecular species within 23 different lipid classes. This enhanced coverage allowed us to identify lipid molecular species and even classes that are altered upon stress, allowing hypotheses on role of glycosylinositolphosphoceramides (GIPC), steryl glycosides (SG) and acylated steryl glycosides (ASG) in drought stress to be addressed and confirming the findings from numerous previous studies with a single, wide‐ranging lipidomics approach. This extended our knowledge on membrane remodeling during the drought response, integrating sphingolipids and sterol lipids into the current glycerolipid‐based model.     

Phytosphingosine ceramide mainly localizes in the central layer of the unique lamellar phase of skin lipid model systems

Understanding the lipid arrangement within the skin’s outermost layer, the stratum corneum (SC), is important for advancing knowledge on the skin barrier function. The SC lipid matrix consists of ceramides (CERs), cholesterol, and free fatty acids, which form unique crystalline lamellar phases, referred to as the long periodicity phase (LPP) and short periodicity phases. As the SC lipid composition is complex, lipid model systems that mimic the properties of native SC are used to study the SC lipid organization and molecular arrangement. In previous studies, such lipid models were used to determine the molecular organization in the trilayer structure of the LPP unit cell. The aim of this study was to examine the location of CER N-(tetracosanoyl)-phytosphingosine (CER NP) in the unit cell of this lamellar phase and compare its position with CER N-(tetracosanoyl)-sphingosine (CER NS). We selected CER NP as it is the most prevalent CER subclass in the human SC, and its location in the LPP is not known. Our neutron diffraction results demonstrate that the acyl chain of CER NP was positioned in the central part of the trilayer structure, with a fraction also present in the outer layers, the same location as determined for the acyl chain of CER NS. In addition, our Fourier transformed infrared spectroscopy results are in agreement with this molecular arrangement, suggesting a linear arrangement for the CER NS and CER NP. These findings provide more detailed insight into the lipid organization in the SC lipid matrix.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

Combined LC/MS-platform for analysis of all major stratum corneum lipids,and the profiling of skin substitutes

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10 μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, ‘high-throughput’ profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3 months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.     

Lipidomics in biomedical research-practical considerations

Lipids have many central physiological roles including as structural components of cell membranes, energy storage sources and intermediates in signaling pathways. Lipid-related disturbances are known to underlie many diseases and their co-morbidities. The emergence of lipidomics has empowered researchers to study lipid metabolism at the cellular as well as physiological levels at a greater depth than was previously possible. The key challenges ahead in the field of lipidomics in medical research lie in the development of experimental protocols and in silico techniques needed to study lipidomes at the systems level. Clinical questions where lipidomics may have an impact in healthcare settings also need to be identified, both from the health outcomes and health economics perspectives. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

Alterations in Mouse Brain Lipidome after Disruption of CST Gene: A Lipidomics Study

To investigate the effects of a critical enzyme, cerebroside sulfotransferase (CST), involving sulfatide biosynthesis on lipid (particularly sphingolipid) homeostasis, herein, we determined the lipidomes of brain cortex and spinal cord from CST null and heterozygous (CST^?/? and CST^+/?, respectively) mice in comparison to their wild-type littermates by multi-dimensional mass spectrometry-based shotgun lipidomics. As anticipated, we demonstrated the absence of sulfatide in the tissues from CST^?/? mice and found that significant reduction of sulfatide mass levels was also present, but in an age-dependent manner, in CST^+/? mice. Unexpectedly, we revealed that the profiles of sulfatide species in CST^+/? mice were significantly different from that of littermate controls with an increase in the composition of species containing saturated and hydroxylated fatty acyl chains. Contrary to the changes of sulfatide levels, shotgun lipidomics analysis did not detect significant changes of the mass levels of other lipid classes examined. Taken together, shotgun lipidomics analysis demonstrated anticipated sulfatide mass deficiency in CST defect mouse brain and revealed novel brain lipidome homeostasis in these mice. These results might provide new insights into the role of CST in myelin function.     

Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics

By using shotgun lipidomics based on the separation of lipid classes in the electrospray ion source (intrasource separation) and two-dimensional (2D) MS techniques (Han, X., and R. W. Gross. 2004. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. First published on June 18, 2004; doi: 10.1002/mas.20023, In press), individual molecular species of most major and many minor lipid classes can be quantitated directly from biological lipid extracts. Herein, we extended shotgun lipidomics to the characterization and quantitation of cerebroside molecular species in biological samples. By exploiting the differential fragmentation patterns of chlorine adducts using electrospray ionization (ESI) tandem mass spectrometry, hydroxy and nonhydroxy cerebroside species are readily identified. The hexose (either galactose or glucose) moiety of a cerebroside species can be distinguished by examination of the peak intensity ratio of its product ions at m/z 179 and 89 (i.e., 0.74 +/- 0.10 and 4.8 +/- 0.7 for galactose- and glucose-containing cerebroside species, respectively). Quantitation of cerebroside molecular species (as little as 10 fmol) from chloroform extracts of brain tissue samples was directly conducted by 2D ESI/MS after correction for differences in (13)C-isotopomer intensities. This method was demonstrated to have a greater than 1,000-fold linear dynamic range in the low concentration region; therefore, it should have a wide range of applications in studies of the cellular sphingolipid lipidome.     

Informatics and computational strategies for the study of lipids

The ability to translate vast amounts of information, as obtained from lipidomic analysis, into the knowledge and understanding of biological phenomena is an important challenge faced by the lipidomics community. While many of the informatics and computational tools from other domains such as bioinformatics and metabolomics are also applicable to lipidomics data processing and analysis, new solutions and strategies are needed for the studies of lipidomes at the systems level. This is due to enormous functional and structural diversity of lipids as well as because of their complex regulation at multiple spatial and temporal scales. In order to better understand the lipidomes at the physiological level, lipids need to be modeled not only at the level of biological pathways but also at the level of the biophysical systems they are part of, such as cellular membranes or lipoprotein particles. Herein the current state, recent advances and new opportunities in the field of lipid bioinformatics are reviewed.     

Net CO2 exchange of Pinus taeda shoots exposed to variable ozone levels and rain chemistries in field and laboratory settings

Net CO₂ exchange rates (CERs) were measured in seedlings of two loblotly pine ( Pinus taeda L.) families following 6- or 13-week exposures to ozone (charcoalfiltered or ambient air + O₃) and acid rain treatments (pH 3.3, 4.5 and 5.2). Ozone exposures (14 or 170 nl l⁻¹) were made in open-top chambers, and in continously stirred tank reactors (14, 160 or 320 nl l⁻¹) located in the field and laboratory, respectively. The CERs of whole shoots were measured in an open infrared gas analysis system at 6 levels of photosynthetic photon flux density (0, 33, 60, 410, 800 and 1660 μmol m⁻² s⁻¹). Treatment effects were not consistent between field- and laboratory-exposed seedlings. Ozone-treated field seedlings exhibited statistically significant reductions in light-saturated CER of 12.5 and 25% when measured at 6 and 13 weeks, respectively. Laboratory seedlings exhibited mixed responses to O₃, with one family showing reduced CER only after 6 weeks of O₃ exposure and the other only after 13 weeks (O₃ >160 nl l⁻¹ for both). After 13 weeks of exposure, pH 3.3, and 4.5 rain treatments enhanced light-saturated CER by an average of 52% over that observed in seedlings exposed to the pH 5.2 treatment. Enhanced CERs due to acid rain were of the same magnitude (3–5 μmol CO₂g⁻¹ s⁻¹) as ozone-induced CER reductions. No differences in dark respiration were detected between treatments. Although ozone and acid rain treatments altered seedling CER, the differences were not translated into altered final plant dry weights over the 13-week exposure period.