Thoracic canal morphology on preoperative magnetic resonance imaging in spinal cord stimulation patients.

INTRODUCTION: In high-frequency spinal cord stimulation anatomic placement targeting of the T9-10 disc space is based on "empiric" results that are best replicated with coverage broadly from T8 to T10. This study contains the largest cohort of patients evaluating low thoracic morphology and seeks to address the lack of MRI morphological analysis in literature. METHODS: This study was a retrospective review of a database of 101 consecutive patients undergoing permanent implant of thoracic SCS for chronic pain. Measurements were carried out on preoperative MRI imaging. Anteroposterior (AP) and lateral dimensions of the spinal cord as well as dural sac were measured. In addition, dorsal cerebrospinal fluid thickness and paddle depression distance were also measured. RESULTS: When comparing morphological dimensions by level, dorsal CSF thickness was smaller at T9-10 than T7-8 (p = 0.018). In addition, lateral dural and spinal cord diameters were larger at T10-11 than T9-10, contributing to larger dural surface area at T10-11 (p = 0.028). While trends of dorsal CSF thickness tend to decrease with lower thoracic levels, the ratio of surface area of spinal cord to dural sac appeared to remain relatively constant. CONCLUSIONS: Dorsal CSF thickness is smaller at T9-10 than T7-8 in chronic pain patients in this cohort. More ellipsoid, cord, and spinal canal diameter measurements were noted at lower levels of the thoracic spinal cord, particularly at T10-11. This may correlate with anatomical SCS placement. Future studies should evaluate efficacy of SCS therapy for pain based on these anatomical considerations.

A jasmonic acid derivative improves skin healing and induces changes in proteoglycan expression and glycosaminoglycan structure.

BACKGROUND: Jasmonates are plant hormones that exhibit anti-cancer and anti-inflammatory properties and have therefore raised interest for human health applications. The molecular basis of these activities remains poorly understood, although increasing evidence suggests that a variety of mechanisms may be involved. Recently, we have reported that a jasmonate derivative (JAD) displayed anti-aging effects on human skin by inducing extracellular matrix (ECM) remodeling. Based on this observation, we have investigated here the effects of JAD on proteoglycans and glycosaminoglycan (GAG) polysaccharides, which are major cell-surface/ECM components and are involved in a multitude of biological processes. In parallel, we have examined the ability of JAD to promote growth factor activities and improve skin wound healing. METHODS: Proteoglycan expression was analyzed on epidermal primary keratinocytes and reconstituted skin epidermis, using electron/immunofluorescence microscopy, western blotting and flow cytometry. GAG composition was determined by disaccharide analysis. Finally, biological activities of JAD were assessed in cellulo, in FGF-7 induced migration/proliferation assays, as well as in vivo, using a suction blister model performed on 24 healthy volunteers. RESULTS: JAD was found to induce expression of major skin proteoglycans and to induce subtle changes in GAG structure. In parallel, we showed that JAD promoted FGF-7 and improved skin healing by accelerating epithelial repair in vivo. CONCLUSION: This study highlights JAD as a promising compound for investigating GAG structure-function relationships and for applications in skin cosmetic /corrective strategies. GENERAL SIGNIFICANCE: We propose here a novel mechanism, by which jasmonate derivatives may elicit biological activities in mammals.

Effect of Different Crosslinking Technologies on Hyaluronic Acid Behavior: A Visual and Microscopic Study of Seven Hyaluronic Acid Gels.

BACKGROUND: The mechanical, rheological, and pharmacological properties of hyaluronic acid (HA) gels differ by their proprietary crosslinking technologies.
OBJECTIVE: To examine the different properties of a range of HA gels using simple and easily reproducible laboratory tests to better understand their suitability for particular indications.
METHODS AND MATERIALS: Hyaluronic acid gels produced by one of 7 different crosslinking technologies were subjected to tests for cohesivity, resistance to stretch, and microscopic examination. These 7 gels were: non-animal stabilized HA (NASHA® [Restylane®]), 3D Matrix (Surgiderm® 24 XP), cohesive polydensified matrix (CPM® [Belotero® Balance]), interpenetrating network-like (IPN-like [Stylage® M]), Vycross® (Juvéderm Volbella®), optimal balance technology (OBT® [Emervel Classic]), and resilient HA (RHA® [Teosyal Global Action]).
RESULTS: Cohesivity varied for the 7 gels, with NASHA being the least cohesive and CPM the most cohesive. The remaining gels could be described as partially cohesive. The resistance to stretch test confirmed the cohesivity findings, with CPM having the greatest resistance. Light microscopy of the 7 gels revealed HA particles of varying size and distribution. CPM was the only gel to have no particles visible at a microscopic level.
CONCLUSION: Hyaluronic acid gels are produced with a range of different crosslinking technologies. Simple laboratory tests show how these can influence a gel's behavior, and can help physicians select the optimal product for a specific treatment indication.

Versions of this paper have been previously published in French and in Dutch in the Belgian journal Dermatologie Actualité. Micheels P, Sarazin D, Tran C, Salomon D. Un gel d'acide hyaluronique est-il semblable à son concurrent? Derm-Actu. 2015;14:38-43.

J Drugs Dermatol. 2016;15(5):600-606..

In vivo bio-integration of three hyaluronic acid fillers in human skin: a histological study.

BACKGROUND: Hyaluronic acid (HA) formulations are used for aesthetic applications. Different cross-linking technologies result in HA dermal fillers with specific characteristic visco-elastic properties. OBJECTIVE: Bio-integration of three CE-marked HA dermal fillers, a cohesive (monophasic) polydensified, a cohesive (monophasic) monodensified and a non-cohesive (biphasic) filler, was analysed with a follow-up of 114 days after injection. Our aim was to study the tolerability and inflammatory response of these fillers, their patterns of distribution in the dermis, and influence on tissue integrity. METHODS: Three HA formulations were injected intradermally into the iliac crest region in 15 subjects. Tissue samples were analysed after 8 and 114 days by histology and immunohistochemistry, and visualized using optical and transmission electron microscopy. RESULTS: Histological results demonstrated that the tested HA fillers showed specific characteristic bio-integration patterns in the reticular dermis. Observations under the optical and electron microscopes revealed morphological conservation of cutaneous structures. Immunohistochemical results confirmed absence of inflammation, immune response and granuloma. CONCLUSION: The three tested dermal fillers show an excellent tolerability and preservation of the dermal cells and matrix components. Their tissue integration was dependent on their visco-elastic properties. The cohesive polydensified filler showed the most homogeneous integration with an optimal spreading within the reticular dermis, which is achieved by filling even the smallest spaces between collagen bundles and elastin fibrils, while preserving the structural integrity of the latter. Absence of adverse reactions confirms safety of the tested HA dermal fillers.

Estimated Impact of Low Isolate Numbers on the Reliability of Cumulative Antibiogram Data.

Antibiograms are cumulative reports of antimicrobial susceptibility results that are used to guide the selection of empirical antibiotic therapy. Although Clinical and Laboratory Standards Institute (CLSI) guidelines recommend including only organisms that have at least 30 isolates in an antibiogram, previous studies demonstrated that adherence to this recommendation is highly variable. This paper aims to model the impact of small sample sizes on expected levels of error in cumulative antibiograms by comparing percent susceptibility results for random samples to those of the larger, entire data set. The results demonstrate relatively high error rates when utilizing low numbers of isolates in cumulative antibiograms, and provide a discussion point for considering the appropriate number of isolates that could be utilized, and the impact of increasing isolate numbers by including multiple years of data. IMPORTANCE Antibiograms are reports of local antimicrobial susceptibility patterns for common bacteria and yeast that are used to make empirical decisions for patient therapy and also to inform institution therapy guidelines. This study evaluates the impact of low isolate counts on the reliability of antibiograms, and suggests that more institutions should utilize multiple years of data to overcome this issue.

Hyaluronic acid skin penetration evaluated by tape stripping using ELISA kit assay.

Hyaluronic Acid (HA) is an endogenous skin matrix component with moisturizing and anti-inflammatory and healing properties. Cosmetic formulations containing HA aim to enhance skin structure, hydrate skin and reduce wrinkles. Therefore, the skin diffusion of HA into stratum corneum after application of a formulation containing two different size of HA, High Molecular weight (HMW-HA) and Low Molecular Weight (LMW-HA)) was evaluated. Ex vivo human skin samples were used to validate an ELISA assay measuring HA in the stratum corneum (SC), viable epidermis and dermis, and to identify optimal washing and extraction methods. These methods were used to measure HA levels in the SC of subjects before and after daily topical application of an HA-containing formulation for 7 days. Samples of SC (5 tape strips) were taken before and 2 h after the application on D0, D1 and D7. The ELISA assay was suitable for measuring HA in the SC but not epidermal or dermal layers. The upper and lower limits of quantitation were the same for both sizes of HA (200 and 3.1 ng/ml, respectively). In both ex vivo human skin and human volunteers, the "dry method" of removing the formulation led to much higher levels of HA in the SC samples, whereas the "wet method" involving one cotton swab soaked with an aqueous solution containing 10% soap and a second cotton swab for drying, was effective in removing the formulation and more relevant to simulate washing/showering. In the clinical study, the amount of HA in SC layers 3-5 were used to represent the HA level in the SC, whereas layers 1 and 2 were considered as surface "residual film". After each application, there was a significantly higher amount of HA compared to the amount before application, which was observed using both wash methods. The residual level 24 h after the first application was at least 8 times higher than before the first application and at least 31 times higher after 7 applications. In conclusion, these investigations validated the use of the ELISA method for the measurement of HA in SC samples. The ex vivo experiments provided recommendations for the clinical study, including the preferred cleansing and optimal sampling methods. The clinical study demonstrated the diffusion, accumulation and maintenance of HA levels in the SC after repeated application of the formulation containing HMW-HA and LMW-HA.

Effects of mercury, organic carbon, and microbial inhibition on methylmercury cycling at the profundal sediment-water interface of a sulfate-rich hypereutrophic reservoir.

Methylmercury (MeHg) produced by anaerobic bacteria in lakes and reservoirs, poses a threat to ecosystem and human health due to its ability to bioaccumulate in aquatic food webs. This study used 48-hr microcosm incubations of profundal sediment and bottom water from a sulfate-rich, hypereutrophic reservoir to assess seasonal patterns of MeHg cycling under various treatments. Treatments included addition of air, Hg(II), organic carbon, and microbial inhibitors. Both aeration and sodium molybdate, a sulfate-reducing bacteria (SRB) inhibitor, generally decreased MeHg concentration in microcosm water, likely by inhibiting SRB activity. The methanogenic inhibitor bromoethanesulfonate increased MeHg concentration 2- to 4- fold, suggesting that methanogens were potent demethylators. Pyruvate increased MeHg concentration under moderately reduced conditions, likely by stimulating SRB, but decreased it under highly reduced conditions, likely by stimulating methanogens. Acetate increased MeHg concentration, likely due to the stimulation of acetotrophic SRB. Results suggest that iron-reducing bacteria (IRB) were not especially prominent methylators and MeHg production at the sediment-water interface is elevated under moderately reduced conditions corresponding with SRB activity. In contrast, it is suppressed under oxic conditions due to low SRB activity, and under highly reduced conditions (<-100 mV) due to enhanced demethylation by methanogens.

Vitamin C Prevents Ultraviolet-induced Pigmentation in Healthy Volunteers: Bayesian Meta-analysis Results from 31 Randomized Controlled versus Vehicle Clinical Studies.

Background: Repeated nonextreme sun exposures induce skin pigmentation by increasing melanin production and by oxidizing preexisting melanin and melanin precursors. This leads to skin disorders and skin color heterogeneity such as hyperpigmented spots. Objective: We assessed 31 randomized, controlled clinical trials to determine the potential of vitamin C to limit ultraviolet (UV) daylight-induced pigmentation, considering dose response and different skin type populations (Caucasian and Chinese). Materials and Methods: Thirty-one intraindividual, randomized, controlled clinical trials involving Caucasian and Chinese subjects (15-35 healthy male or female volunteers per study, 741 total volunteers) 18 to 50 years of age with Phototype III and individual typology angle (ITA) value between 28 and 49 degrees were analyzed. The 31 studies assessed the potential of vitamin C (formulated with the copolymer Styrène-Anhydride Maléique [SMA]) to decrease pigmentation induced by UV daylight exposure. Results were combined using a Bayesian meta-analysis to provide probabilistic evidence of the effects of vitamin C by dose and population. Results: Vitamin C was effective in reducing pigmentation induced by UV daylight-simulated expositions (4 days at 0.75 Individual Minimal Erythemal Dose [MEDi]) in a dose-dependent manner. During the depigmentation phase, no additive value was provided by the vitamin C, suggesting that the lightening properties described in the literature for vitamin C correspond to an antipigmenting quality rather than a depigmenting effect. Conclusion: Vitamin C is a valuable and safe dermocosmetic antipigmenting compound with a strong effect at 10% possibly useful in preventing signs of photoaging.

Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism.

BACKGROUND: Skin atrophy is a common manifestation of aging and is frequently accompanied by ulceration and delayed wound healing. With an increasingly aging patient population, management of skin atrophy is becoming a major challenge in the clinic, particularly in light of the fact that there are no effective therapeutic options at present. METHODS AND FINDINGS: Atrophic skin displays a decreased hyaluronate (HA) content and expression of the major cell-surface hyaluronate receptor, CD44. In an effort to develop a therapeutic strategy for skin atrophy, we addressed the effect of topical administration of defined-size HA fragments (HAF) on skin trophicity. Treatment of primary keratinocyte cultures with intermediate-size HAF (HAFi; 50,000-400,000 Da) but not with small-size HAF (HAFs; <50,000 Da) or large-size HAF (HAFl; >400,000 Da) induced wild-type (wt) but not CD44-deficient (CD44-/-) keratinocyte proliferation. Topical application of HAFi caused marked epidermal hyperplasia in wt but not in CD44-/- mice, and significant skin thickening in patients with age- or corticosteroid-related skin atrophy. The effect of HAFi on keratinocyte proliferation was abrogated by antibodies against heparin-binding epidermal growth factor (HB-EGF) and its receptor, erbB1, which form a complex with a particular isoform of CD44 (CD44v3), and by tissue inhibitor of metalloproteinase-3 (TIMP-3). CONCLUSIONS: Our observations provide a novel CD44-dependent mechanism for HA oligosaccharide-induced keratinocyte proliferation and suggest that topical HAFi application may provide an attractive therapeutic option in human skin atrophy.

UVA and UVB decrease the expression of CD44 and hyaluronate in mouse epidermis, which is counteracted by topical retinoids.

The transmembrane glycoprotein CD44 is currently thought to be the main cell surface receptor for the glycosaminoglycan hyaluronate. We previously showed that (1) CD44 regulate keratinocyte proliferation; (2) topical retinoids dramatically increase the expression of CD44, hyaluronate and hyaluronate synthase (HAS)s in mouse epidermis; (3) topical retinaldehyde restores the epidermal thickness and CD44 expression which are correlated with clinical improvement in lichen sclerosus et atrophicus lesions; and (4) retinaldehyde-induced proliferative response of keratinocytes is a CD44-dependent phenomenon and requires the presence of HB-EGF, erbB1 and matrix metalloproteinases. In this study, we analyzed the effect of UV irradiation on the levels of epidermal hyaluronate and CD44 in mice, as well as its potential prevention by topical retinoids. UVA (10 J/cm(2)) or UVB (1 J/cm(2)) irradiation significantly decreased the expression of CD44 and hyaluronate in the epidermis of hairless mice after 2 h. Expression of both epidermal CD44 and hyaluronate was reconstituted within 24 h. Topical application of retinaldehyde for 3 days prior to UVA or UVB irradiation prevented the decrease of CD44 and hyaluronate expression. Topical retinol and retinoic acid also increased the basal levels of epidermal CD44 and hyaluronate, although their preventive effect on UV-induced decrease of these molecules was less pronounced as compared to topical retinaldehyde. These data confirm the relationships between retinoid and CD44 pathways, although the primary target(s) of UV leading to CD44 and hyaluronate degradation remain to be elucidated.

Oxidative stress-independent depletion of epidermal vitamin A by UVA.

In hairless mice, epidermal vitamin A (retinol and retinyl esters) is strongly decreased following a single exposure to UVB. Here, using the same mouse model, we studied the effects of UVA on epidermal vitamin A content, lipid peroxidation, and CRBP-I expression, as well as the putative prevention of vitamin A depletion or lipid peroxidation by topical alpha-tocopherol. An acute exposure to UVA completely depleted epidermal vitamin A with EC50 of 0.25 and 0.5 J per cm2 for retinyl esters and retinol, respectively; these values were 0.1 J per cm2 for both retinoids under UVB exposure. CRBP-I expression was increased 2-fold 8 h following UVA exposure (10 J per cm2), and this increase persisted for at least 16 h. A single UVA exposure induced a concentration-dependent epidermal lipid peroxidation (EC50 = 3.5 J per cm2) giving rise to 55.4 +/- 4.2 nmol lipid peroxides per g at 20 J per cm2, whereas UVB, up to 1 J per cm2, did not increase the basal concentration of 6.7 +/- 0.9 nmol lipid peroxides per g. On the other hand, topical menadione induced a concentration-dependent lipid peroxidation, but did not affect vitamin A content. Pretreatment with alpha-tocopherol (i) did not inhibit UV-induced vitamin A depletion, (ii) completely inhibited the increased lipid peroxidation induced by UVA or menadione, and (iii) accelerated reconstitution of epidermal vitamin A after UVB but not UVA induced depletion. Thus acute UVA induced both epidermal vitamin A depletion and lipid peroxidation, UVB induced only vitamin A depletion, and menadione induced only a lipid peroxidation; topical alpha-tocopherol prevented lipid peroxidation but not vitamin A depletion. These observations indicate (i) that CRBP-I neither provides protection to UVB- and UVA-induced epidermal vitamin A depletion, nor interferes significantly with reconstitution, and (ii) that the UV-induced vitamin A depletion and lipid peroxidation in mouse epidermis are unrelated processes. UV light does not destroy epidermal vitamin A through an oxidative stress but probably by a photochemical reaction in which UV radiations at about 325 nm give the corresponding activation energy.

Vitamin A exerts a photoprotective action in skin by absorbing ultraviolet B radiation.

Retinyl esters, a storage form of vitamin A, concentrate in the epidermis, and absorb ultraviolet radiation with a maximum at 325 nm. We wondered whether these absorbing properties of retinyl esters might have a biologically relevant filter activity. We first used an in vitro model to assess the photoprotective properties of retinyl palmitate. We then applied topical retinyl palmitate on the back of hairless mice before exposing them to 1 J per cm2 ultraviolet B, and assayed the levels of thymine dimers produced in epidermal DNA 2 h following ultraviolet B exposure. Finally, we applied topical retinyl palmitate or a sunscreen on the buttocks of human volunteers before exposing them to four minimal erythema doses of ultraviolet B; we assayed the levels of thymine dimers produced 2 h following ultraviolet B exposure, and determined the intensity of erythema 24 h after ultraviolet B. In vitro, retinyl palmitate was shown to be as efficient as the commercial filter octylmethoxycinnamate in preventing ultraviolet-induced fluorescence or photobleaching of fluorescent markers. The formation of thymine dimers in mouse epidermis was significantly inhibited by topical retinyl palmitate. In human subjects, topical retinyl palmitate was as efficient as a sun protection factor 20 sunscreen in preventing sunburn erythema as well as the formation of thymine dimers. These results demonstrate that epidermal retinyl esters have a biologically relevant filter activity and suggest, besides their pleomorphic biologic actions, a new role for vitamin A that concentrates in the epidermis.

A new model using liposomes that allow to distinguish between absorption and oxidative properties of sunscreens.

We have developed a new model using liposome-encapsulated fluorescent probes, aiming at assessing both the physical and the biological protection provided by filter molecules such as those incorporated in sunscreens. The fluorescent indicator Indo-1 or 2',7'-dichlorofluorescin (DCFH) was inside the liposomes, in the aqueous inner compartment, whereas the filter molecules octyl methoxycinnamate (OMC), benzophenone-3 (BP3) or avobenzone, widely used in sunscreens, were incorporated into liposome membranes. When liposome suspensions were placed in a fluorometer cuvette exposed to an incident UV beam, the decrease of Indo-1 fluorescence as a function of filter concentration was related to the extinction coefficient of the filters. On the other hand, when liposome suspensions were exposed to moderate UVB doses allowing Indo-1 photobleaching, the remaining intact Indo-1 was linked to the protection provided by filter-containing liposome membranes. Finally, when liposome-encapsulated DCFH was exposed to UVB, the degree of photo-oxidation of the fluorescent probe into 2',7'-dichlorofluorescein accounted for the photoprotection provided by the filter contained in liposome membranes. BP3 was more potent and slightly less efficient than the other two filters in preventing Indo-1 fluorescence; all three filters provided a similar concentration-dependent protection of Indo-1 photobleaching, whereas only OMC was able to prevent the photooxidation of DCFH. The liposome model presented here has the advantage of combining both physical and biological parameters to assess the photoprotection provided by filter molecules, and the lack of photoprotection by two sunscreen molecules having a good filter capacity highlights the need for such a biological parameter when talking about the safety of sunscreens.

Synergistic effect of hyaluronate fragments in retinaldehyde-induced skin hyperplasia which is a Cd44-dependent phenomenon.

BACKGROUND: CD44 is a polymorphic proteoglycan and functions as the principal cell-surface receptor for hyaluronate (HA). Heparin-binding epidermal growth factor (HB-EGF) activation of keratinocyte erbB receptors has been proposed to mediate retinoid-induced epidermal hyperplasia. We have recently shown that intermediate size HA fragments (HAFi) reverse skin atrophy by a CD44-dependent mechanism. METHODOLOGY AND PRINCIPAL FINDINGS: Treatment of primary mouse keratinocyte cultures with retinaldehyde (RAL) resulted in the most significant increase in keratinocyte proliferation when compared with other retinoids, retinoic acid, retinol or retinoyl palmitate. RAL and HAFi showed a more significant increase in keratinocyte proliferation than RAL or HAFi alone. No proliferation with RAL was observed in CD44-/- keratinocytes. HA synthesis inhibitor, 4-methylumbelliferone inhibited the proliferative effect of RAL. HB-EGF, erbB1, and tissue inhibitor of MMP-3 blocking antibodies abrogated the RAL- or RAL- and HAFi-induced keratinocyte proliferation. Topical application of RAL or RAL and HAFi for 3 days caused a significant epidermal hyperplasia in the back skin of wild-type mice but not in CD44-/- mice. Topical RAL and HAFi increased epidermal CD44 expression, and the epidermal and dermal HA. RAL induced the expression of active HB-EGF and erbB1. However, treatment with RAL and HAFi showed a more significant increase in pro-HB-EGF when compared to RAL or HAFi treatments alone. We then topically applied RAL and HAFi twice a day to the forearm skin of elderly dermatoporosis patients. After 1 month of treatment, we observed a significant clinical improvement. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that (i) RAL-induced in vitro and in vivo keratinocyte proliferation is a CD44-dependent phenomenon and requires the presence of HA, HB-EGF, erbB1 and MMPs, (ii) RAL and HAFi show a synergy in vitro and in vivo in mouse skin, and (iii) the combination of RAL and HAFi seems to have an important therapeutic effect in dermatoporosis.

Metabolism and biological activities of topical 4-oxoretinoids in mouse skin.

Retinoic acid mediates most of the biological actions of vitamin A. It is oxidized by CYP26A1 to 4-oxoretinoic acid, considered as an inactive catabolite of retinoic acid. However, in the light of studies reporting the presence of 4-oxoretinal or 4-oxoretinol as the predominant retinoids during morphogenesis, we analyzed the retinoid-like biological activity of these oxoretinoids in mouse skin in vivo. Topical 4-oxoretinal and 4-oxoretinol promoted significant epidermal hyperplasia and metaplasia in mouse tail. They induced a moderate response for epidermal inflammation, compared with retinal, whereas neither 4-oxoretinal nor 4-oxoretinol prevented menadione-induced epidermal lipid peroxidation, unlike retinal and retinol. As analyzed by quantitative PCR, 4-oxoretinal and 4-oxoretinol did not reproduce the significant increased expression of genes coding for keratin 4, amphiregulin, heparin-EGF and CYP26A1, that did induce retinal and retinol. However, both retinal and 4-oxoretinal significantly inhibited the lipopolysaccharide-induced maturation of human dendritic cells in vitro. As analyzed in vivo and in vitro, 4-oxoretinal and 4-oxoretinol were not converted into retinoic acid. We conclude that 4-oxoretinal and 4-oxoretinol exert a moderate direct retinoid-like activity in vivo, thus confirming previous in vitro studies in amphibians showing 4-oxometabolites of vitamin A as bioactive agents rather than inactive catabolites.