AlphaFold accelerated discovery of psychotropic agonists targeting the trace amine-associated receptor 1.

Artificial intelligence is revolutionizing protein structure prediction, providing unprecedented opportunities for drug design. To assess the potential impact on ligand discovery, we compared virtual screens using protein structures generated by the AlphaFold machine learning method and traditional homology modeling. More than 16 million compounds were docked to models of the trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor of unknown structure and target for treating neuropsychiatric disorders. Sets of 30 and 32 highly ranked compounds from the AlphaFold and homology model screens, respectively, were experimentally evaluated. Of these, 25 were TAAR1 agonists with potencies ranging from 12 to 0.03 µM. The AlphaFold screen yielded a more than twofold higher hit rate (60%) than the homology model and discovered the most potent agonists. A TAAR1 agonist with a promising selectivity profile and drug-like properties showed physiological and antipsychotic-like effects in wild-type but not in TAAR1 knockout mice. These results demonstrate that AlphaFold structures can accelerate drug discovery.

Transfer of cetirizine/levocetirizine into human breast milk and estimation of drug exposure to infants through breastfeeding: A human lactation study from the ConcePTION project.

Data on drug transfer into human breast milk are sparse. This study aimed to quantify concentrations of cetirizine and levocetirizine in breast milk and to estimate drug exposure to infants. Breastfeeding women at least 8 weeks postpartum and using cetirizine or its pure (R)-enantiomer levocetirizine were eligible to participate. Breast milk samples were collected at six predefined times during a dose interval (0, 2, 4, 8, 12 and 24 h after drug intake) at steady state. Infant drug exposure was estimated by calculating the absolute infant dose (AID) and the weight-adjusted relative infant dose (RID). In total, 32 women were eligible for final inclusion, 31 women using cetirizine and one woman using levocetirizine. Means of the individual maximum and average cetirizine milk concentrations were 41.0 and 16.8 µg/L, respectively. Maximum concentrations occurred on average 2.4 h after intake, and the mean half-life in milk was 7.0 h. Estimated AID and RID for cetirizine in a day were 2.5 µg/kg and 1.9%, respectively. The corresponding values for levocetirizine were 1.1 µg/kg and 1.9%. No severe adverse events were reported. Our findings demonstrate that the transfer of cetirizine and levocetirizine into breast milk is low and compatible with breastfeeding.

Simple and rapid quantification of cetirizine, venlafaxine, and O-desmethylvenlafaxine in human breast milk, and metformin in human milk and plasma with UHPLC-MS/MS.

The majority of women have health problems that require medication after giving birth. Complications such as allergies, postpartum depression, and diabetes are often treated with drugs such as cetirizine, venlafaxine, and metformin, respectively. These treatments are considered safe during lactation, but information of the transfer of drugs to breast milk and possible effects on the infant is scarce. Therefore, this needs to be systematically investigated in larger populations. To enable the determination of drug transfer, we here describe the validation of two rapid, sensitive, and high-throughput analysis methods for 1) simultaneous quantification of cetirizine, venlafaxine, and O-desmethylvenlafaxine in human breast milk, and 2) metformin in human breast milk and plasma. In both methods, a simple protein precipitation protocol with acetonitrile and benchtop-centrifugation was used prior to compound analysis with liquid-chromatography tandem mass spectrometry. The methods had linear ranges between 0.39 - 194.5 ng/mL for cetirizine, 0.28 - 138.7 ng/mL for venlafaxine, 0.26 - 131.7 ng/mL for O-desmethylvenlafaxine, in milk, and 0.65 - 193.7 ng/mL for metformin in both milk and plasma. Intra-run and inter-run precision and accuracy were ≤ 9% for cetirizine, venlafaxine, and O-desmethylvenlafaxine in milk, and ≤ 7% for metformin in milk and plasma. Cetirizine was measured to median milk concentrations of 13 ng/mL (range: 0.65 - 65 ng/mL) in 228 donor samples from breast-feeding women.

Pharmacokinetic considerations of nanodelivery to the brain: Using modeling and simulations to predict the outcome of liposomal formulations.

The use of nanocarriers is an intriguing solution to increase the brain delivery of novel therapeutics. The aim of this paper was to use pharmacokinetic analysis and simulations to identify key factors that determine the effective drug concentration-time profile at the target site in the brain. Model building and simulations were based on experimental data obtained from the administration of the opioid peptide DAMGO in glutathione tagged PEGylated liposomes to rats. Different pharmacokinetic models were investigated to explore the mechanisms of increased brain delivery. Concentration-time profiles for a set of formulations with varying compound and carrier characteristics were simulated. By controlling the release rate from the liposome, the time profile and the extent of brain delivery can be regulated. The modeling did not support a mechanism of the liposomes passing the brain endothelial cell membrane in an intact form through endocytosis or transcytosis. The most likely process was found to be fusion of the liposome with the endothelial luminal membrane. The simulations revealed that low permeable compounds, independent on efflux, will gain the most from a nanocarrier formulation. The present model based approach is useful to explore and predict possibilities and limitations of carrier-based systems to the brain.

Exploring Factors Causing Low Brain Penetration of the Opioid Peptide DAMGO through Experimental Methods and Modeling.

To advance the development of peptide analogues for improved treatment of pain, we need to learn more about the blood-brain barrier transport of these substances. A low penetration into the brain, with an unbound brain to blood ratio, Kp,uu, of 0.08, is an important reason for the lack of effect of the enkephalin analogue DAMGO (H-Tyr-d-Ala-Gly-MePhe-Gly-ol) according to earlier findings. The aim of this study was to investigate the role of efflux transporters, metabolism in the brain, and/or elimination through interstitial fluid bulk flow for the brain exposure of DAMGO. The in vivo brain distribution of DAMGO was evaluated using microdialysis in the rat. Data were analyzed with population modeling which resulted in a clearance into the brain of 1.1 and an efflux clearance 14 µL/min/g_brain. The efflux clearance was thus much higher than the bulk flow known from the literature. Coadministration with the efflux transporter inhibitors cyclosporin A and elacridar in vivo did not affect Kp,uu. The permeability of DAMGO in the Caco-2 assay was very low, of the same size as mannitol. The efflux ratio was <2 and not influenced by cyclosporin A or elacridar. These results indicate that the well-known efflux transporters Pgp and Bcrp are not responsible for the higher efflux of DAMGO, which opens up for an important role of other transporters at the BBB.

In vivo Functional Evaluation of Increased Brain Delivery of the Opioid Peptide DAMGO by Glutathione-PEGylated Liposomes.

PURPOSE: The purpose of this study was to evaluate formulation factors causing improvement in brain delivery of a small peptide after encapsulation into a targeted nanocarrier in vivo. METHODS: The evaluation was performed in rats using microdialysis, which enabled continuous sampling of the released drug in both the brain (striatum) and blood. Uptake in brain could thereby be studied in terms of therapeutically active, released drug. RESULTS: We found that encapsulation of the peptide DAMGO in fast-releasing polyethylene glycol (PEG)ylated liposomes, either with or without the specific brain targeting ligand glutathione (GSH), doubled the uptake of DAMGO into the rat brain. The increased brain delivery was observed only when the drug was encapsulated into the liposomes, thus excluding any effects of the liposomes themselves on the blood-brain barrier integrity as a possible mechanism. The addition of a GSH coating on the liposomes did not result in an additional increase in DAMGO concentrations in the brain, in contrast to earlier studies on GSH coating. This may be caused by differences in the characteristics of the encapsulated compounds and the composition of the liposome formulations. CONCLUSIONS: We were able to show that encapsulation into PEGylated liposomes of a peptide with limited brain delivery could double the drug uptake into the brain without using a specific brain targeting ligand.

The impact of reference gene selection in quantification of gene expression levels in guinea pig cervical tissues and cells.

BACKGROUND: Accurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix. RESULTS: Gene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes. CONCLUSIONS: We recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells from guinea pig.

Enhanced brain delivery of the opioid peptide DAMGO in glutathione pegylated liposomes: a microdialysis study.

Glutathione PEGylated (GSH-PEG) liposomes were evaluated for their ability to enhance and prolong blood-to-brain drug delivery of the opioid peptide DAMGO (H-Tyr-d-Ala-Gly-MePhe-Gly-ol). An intravenous loading dose of DAMGO followed by a 2 h constant rate infusion was administered to rats, and after a washout period of 1 h, GSH-PEG liposomal DAMGO was administered using a similar dosing regimen. DAMGO and GSH-PEG liposomal DAMGO were also administered as a 10 min infusion to compare the disposition of the two formulations. Microdialysis made it possible to determine free DAMGO in brain and plasma, while the GSH-PEG liposomal encapsulated DAMGO was measured with regular plasma sampling. The antinociceptive effect of DAMGO was determined with the tail-flick method. All samples were analyzed using liquid chromatography-tandem mass spectrometry. The short infusion of DAMGO resulted in a fast decline of the peptide concentration in plasma with a half-life of 9.2 ± 2.1 min. Encapsulation in GSH-PEG liposomes prolonged the half-life to 6.9 ± 2.3 h. Free DAMGO entered the brain to a limited extent with a steady state ratio between unbound drug concentrations in brain interstitial fluid and in blood (Kp,uu) of 0.09 ± 0.04. GSH-PEG liposomes significantly increased the brain exposure of DAMGO to a Kp,uu of 0.21 ± 0.17 (p < 0.05). By monitoring the released, active substance in both blood and brain interstitial fluid over time, we were able to demonstrate that GSH-PEG liposomes offer a promising platform for enhancing and prolonging the delivery of drugs to the brain.

Quantitative analysis of the opioid peptide DAMGO in rat plasma and microdialysis samples using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of the opioid peptide DAMGO in rat plasma, as well as DAMGO and the microdialysis recovery calibrator [(13)C(2),(15)N]-DAMGO in microdialysis samples, is described. The microdialysis samples consisted of 15 µL Ringer solution containing 0.5% bovine serum albumin. Pretreatment of the samples involved protein precipitation with acetonitrile followed by dilution with 0.01% formic acid. The lower limits of quantification were 0.52 ng/mL and 0.24 ng/mL for DAMGO and [(13)C(2),(15)N]-DAMGO respectively and the response was linear up to 5000 fold higher concentrations. The plasma samples (50 µL) were precipitated with acetonitrile containing the isotope labeled analog [(13)C(2),(15)N]-DAMGO as internal standard. The method was linear in the range of 11-110,000 ng/mL. The separations were conducted on a HyPurity C18 column, 50×4.6 mm, 3 µm particle size, with a mobile phase consisting of acetonitrile, water and formic acid to the proportions of 17.5:82.5:0.01. Low energy collision dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 514.2→453.2 for DAMGO and m/z 517.2→456.2 for [(13)C(2),(15)N]-DAMGO. The intra-day precision and accuracy did not exceed 5.2% and 93-104% for both compounds and sample types described. The inter-day precision an accuracy were <6.8% and 95-105% respectively. The method described is simple, reproducible and suitable for the analysis of small sample volumes at low concentrations.

Transforming growth factor ß1 (TGFß1) and progesterone regulate matrix metalloproteinases (MMP) in human endometrial stromal cells.

CONTEXT: Menstruation is preceded by progesterone withdrawal and endometrial matrix remodeling predominantly through induction of matrix metalloproteinases (MMP) and recruitment of invading neutrophils. DESIGN: Using endometrial tissues from women during various phases of the menstrual cycle, we found that MMP2, MMP9, and MMP11 were up-regulated in the late secretory phase/premenstrual phase. Because TGFß-responsive genes were also up-regulated in endometrium during this time, we tested the hypothesis that TGFß1 and progesterone regulate expression of MMP in human endometrial stromal cells (HESC). RESULTS: Treatment of HESC with TGFß1 resulted in marked increases in MMP2 and MMP11 mRNA and pro- and active MMP2 activity. Progesterone inhibited TGFß1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGFß1 also decreased progesterone receptor (PR)-A and PR-B in HESC with a more pronounced effect on PR-A. CONCLUSIONS: These data support the hypothesis that TGFß1 has endogenous anti-progestational effects in HESC and that the opposing effects of progesterone and TGFß1 are important in regulation of matrix integrity in human endometrium.

Health-related quality of life among women with breast cancer - a population-based study.

BACKGROUND: High incidence rates of breast cancer emphasize the importance of increased knowledge about the health-related quality of life (HRQoL) in this patient group. The aim of the present study was to describe and compare HRQoL among breast cancer patients shortly after diagnosis with normative data from the general population, and to investigate how clinical, demographic, and socio-economic factors and social support are associated with HRQoL. MATERIAL AND METHODS: Participants were identified in a population-based Breast Cancer Quality Register in central Sweden. Of 1573 women newly diagnosed with breast cancer during a one-year period (2007-2008), 69% (n = 1086) completed a questionnaire including the EORTC QLQ-C30, BR23 and the HADS. RESULTS: Compared to age-adjusted normative data, breast cancer patients (mean age 62 years, range 25-94), especially younger women (<50 years), experienced clinically meaningful poorer HRQoL. Clinically significant levels of anxiety and depressive symptoms were found among 14% and 6% of the patients, respectively. Factors associated with more problems/symptoms among study participants included chemotherapy, lack of social support, sick leave and a poor financial situation. Adding socio-economic factors diminished the association between age and HRQoL (p > 0.05). CONCLUSION: Recently diagnosed breast cancer patients reported poorer HRQoL in several dimensions compared to normative data. In addition to clinical and demographic factors, an unfavorable socio-economic standing was associated with more problems/symptoms. The present findings emphasize the importance of taking a variety of factors into account when assessing HRQoL in the clinical setting.

17beta-hydroxysteroid dehydrogenase 3 deficiency in a male pseudohermaphrodite.

OBJECTIVE: To present the clinical, biochemical, and genetic features of a male pseudohermaphrodite whose condition was caused by 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) deficiency. DESIGN: Case report. SETTING: Gynecology practice in a university teaching hospital. PATIENT(S): A 15-year-old black American male pseudohermaphrodite with 17beta-HSD3 deficiency. INTERVENTION(S): Laboratory evaluation, genetic mutation analysis, bilateral gonadectomy, and hormone replacement. MAIN OUTCOME MEASURE(S): Endocrinologic evaluation and genetic analysis. RESULT(S): A diagnosis of 17beta-HSD3 deficiency made on the basis of hormone evaluation was confirmed through genetic mutation analysis of the HSD17B3 gene. Female phenotype was attained after gonadectomy, passive vaginal dilatation, and hormone therapy. CONCLUSION(S): Deficiency of 17beta-HSD3 was diagnosed in this patient on the basis of endocrinologic evaluation and was confirmed with genetic mutation analysis. The patient was able to retain her female sexual identity after surgical and medical treatment.

Loss-of-function mutation in carotenoid 15,15'-monooxygenase identified in a patient with hypercarotenemia and hypovitaminosis A.

The enzyme carotenoid 15,15'-monooxygenase (CMO1) catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A in the small intestine. Plant carotenoids are an important dietary source of vitamin A (retinol) and the sole source of vitamin A for vegetarians. Vitamin A is essential for normal embryonic development as well as normal physiological functions in children and adults. Here, we describe one heterozygous T170M missense mutation in the CMO1 gene in a subject with hypercarotenemia and mild hypovitaminosis A. The replacement of a highly conserved threonine with methionine results in a 90% reduction in enzyme activity when analyzed in vitro using purified recombinant enzymes. The Michaelis-Menten constant (K(m)) for the mutated enzyme is normal. Ample amounts of carotenoids are present in plasma of persons consuming a normal Western diet, suggesting that the enzyme is saturated with substrate under normal conditions. Therefore, we propose that haploinsufficiency of the CMO1 enzyme may cause symptoms of hypercarotenemia and hypovitaminosis A in individuals consuming a carotenoid-containing and vitamin A-deficient diet.

Production of recombinant human alpha1-microglobulin and mutant forms involved in chromophore formation.

Alpha(1)-Microglobulin, a 26 kDa lipocalin present in plasma and tissues, carries a set of unknown chromophores, bound to C34, K92, K118 and K130, which cause its charge and size heterogeneity. In man, the protein is found in two forms, full length and lacking the C-terminal tetrapeptide LIPR (t-alpha(1)-microglobulin), both which are heme-binding and the latter with heme-degrading properties. We report cloning and overexpression of full length alpha(1)-microglobulin (wt protein), t-alpha(1)-microglobulin (wtdeltaLIPR) and the mutants C34S, K(92,118,130)T and C34S/K(92,118,130)T, the latter subsequently abbreviated as K(3)T and C34S/K(3)T, in Escherichia coli. After purification and refolding from inclusion bodies, all proteins were correctly folded as determined by far-UV circular dichroism and radioimmunoassay. As revealed by gel filtration, recombinant alpha(1)-microglobulins had lower tendencies to form dimers than human plasma or urine analogues. All alpha(1)-microglobulin forms displayed higher amounts of the chromophore than bovine serum albumin but significantly lower than the human urine or plasma counterparts. Differences in the absorbance and fluorescence profiles are consistent with a model where the chromophore is formed by a series of reactions with heme or other chromophore precursors and where C34 is essential for binding of the ligand, K92, K118 and K130 are involved in transformation into the chromophore and LIPR inhibits the latter reaction.

Deoxycorticosterone inactivation by AKR1C3 in human mineralocorticoid target tissues.

Aldosterone is the principal endogenous mineralocorticoid in humans and regulates salt and water homeostasis. Cortisol, the major glucocorticoid, has high affinity for the mineralocorticoid receptor; however, 11beta-hydroxysteroid dehydrogenase type 2 converts cortisol to the inactive steroid cortisone in aldosterone target cells of the kidney, thus limiting the mineralocorticoid action of cortisol. Deoxycorticosterone (DOC) binds to the mineralocorticocoid receptor with high affinity and circulates at concentrations comparable to aldosterone. Severe DOC excess as is seen in 17alpha- and 11beta-hydroxylase deficiencies causes hypertension, and moderate DOC overproduction in late pregnancy is associated with hypertension. Here, we demonstrate that DOC is inactivated by the 20-ketosteroid reductase activity of the human AKR1C3 isozyme. Immunohistochemical analyses demonstrate that AKR1C3 is expressed in the mineralocorticoid-responsive epithelial cells of the renal cortical and medullary collecting ducts, as well as the colon. Our findings suggest that AKR1C3 protects the mineralocorticoid receptor from activation by DOC in mineralocorticoid target cells of the kidney and colon, analogous to cortisol inactivation by 11beta-hydroxysteroid dehydrogenase type 2.

Cell type-specific expression of beta-carotene 9',10'-monooxygenase in human tissues.

The symmetrically cleaving beta-carotene 15,15'-monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered beta-carotene 9',10'-monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis.

Cell type-specific expression of beta-carotene 15,15'-mono-oxygenase in human tissues.

We studied the cell type-specific expression of human beta-carotene 15,15'-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.

Expression of the AMBP gene transcript and its two protein products, alpha(1)-microglobulin and bikunin, in mouse embryogenesis.

The expression pattern of the alpha(1)-microglobulin/bikunin precursor (AMBP) gene, and its two protein products were studied in mouse embryos of 8.5-15.5 days of embryonic development by in situ hybridization and immunohistochemistry. AMBP mRNA is strongly transcribed in liver parenchyma, pancreas, and intestine epithelium. Sites of weaker expression are the vessels of the umbilical cord, the developing vertebral bodies, and kidney. The alpha(1)-microglobulin and bikunin proteins are accordingly present in developing hepatocytes, pancreas, kidney, and gut. However, additional sites of protein distribution were found that do not correlate to mRNA localization: alpha(1)-microglobulin was found in myocytes and bikunin in cardiac muscle, nervous system microvasculature, and connective tissue. Both proteins were found in brain mesenchyme and meninges. Thus, a restricted expression of the AMBP mRNA in a few organs contrasts to a widespread and unique distribution of each of the two proteins.

Biochemical properties of purified recombinant human beta-carotene 15,15'-monooxygenase.

Beta-carotene 15,15'-monooxygenase (BCO), formerly known as beta-carotene 15,15'-dioxygenase, catalyzes the first step in the synthesis of vitamin A from dietary carotenoids. We have biochemically and enzymologically characterized the purified recombinant human BCO enzyme. A highly active BCO enzyme was expressed and purified to homogeneity from baculovirus-infected Spodoptera frugiperda 9 insect cells. The K(m) and V(max) of the enzyme for beta-carotene were 7 microm and 10 nmol retinal/mg x min, respectively, values that corresponded to a turnover number (k(cat)) of 0.66 min(-1) and a catalytic efficiency (k(cat)/K(m)) of approximately 10(5) m(-1) x min(-1). The enzyme existed as a tetramer in solution, and substrate specificity analyses suggested that at least one unsubstituted beta-ionone ring half-site was imperative for efficient cleavage of the carbon 15,15'-double bond in carotenoid substrates. High levels of BCO mRNA were observed along the whole intestinal tract, in the liver, and in the kidney, whereas lower levels were present in the prostate, testis, ovary, and skeletal muscle. The current data suggest that the human BCO enzyme may, in addition to its well established role in the digestive system, also play a role in peripheral vitamin A synthesis from plasma-borne provitamin A carotenoids.