Vitamin A Abstracts 1

© 2012

Beta-carotene is an important vitamin A source for humans

            (Grune, Lietz et al. 2010) Download

Experts in the field of carotenoids met at the Hohenheim consensus conference in July 2009 to elucidate the current status of beta-carotene research and to summarize the current knowledge with respect to the chemical properties, physiological function, and intake of beta-carotene. The experts discussed 17 questions and reached an agreement formulated in a consensus answer in each case. These consensus answers are based on published valid data, which were carefully reviewed by the individual experts and are justified here by background statements. Ascertaining the impact of beta-carotene on the total dietary intake of vitamin A is complicated, because the efficiency of conversion of beta-carotene to retinol is not a single ratio and different conversion factors have been used in various surveys and following governmental recommendations within different countries. However, a role of beta-carotene in fulfilling the recommended intake for vitamin A is apparent from a variety of studies. Thus, besides elucidating the various functions, distribution, and uptake of beta-carotene, the consensus conference placed special emphasis on the provitamin A function of beta-carotene and the role of beta-carotene in the realization of the required/recommended total vitamin A intake in both developed and developing countries. There was consensus that beta-carotene is a safe source of vitamin A and that the provitamin A function of beta-carotene contributes to vitamin A intake.

Molecular and dietary regulation of beta,beta-carotene 15,15'-monooxygenase 1 (BCMO1)

            (Lietz, Lange et al. 2010) Download

beta,beta-Carotene 15,15'-monooxygenase-1 (BCMO1) is a key enzyme in vitamin A metabolism in mammals. Various dietary components such as non-pro-vitamin A carotenoids, fat, and polyphenols have been shown to influence the intestinal absorption and conversion of pro-vitamin A carotenoids. Furthermore, vitamin A deficiency has been shown to induce BCMO1 expression, whereas supplementation with vitamin A or its active metabolites, all-trans and 9-cis retinoic acid, dose-dependently reverse these effects. A diet-responsive regulatory network involving the intestine specific homeodomain transcription factor ISX has been shown to regulate the intestinal vitamin A uptake and production via a negative feedback control. Furthermore, non-synonymous single nucleotide polymorphisms in the human BCMO1 gene have been discovered causing observably reduced BCMO1 activity. Detailed knowledge about BCMO1 regulation as well as genetic variations causing variable cleavage activities may provide a background, on which individual and/or population based dietary recommendations for beta-carotene and vitamin A intake could be established.

Single nucleotide polymorphisms upstream from the beta-carotene 15,15'-monoxygenase gene influence provitamin A conversion efficiency in female volunteers

            (Lietz, Oxley et al. 2012) Download

beta-Carotene, the most abundant provitamin A carotenoid in the diet, is converted to retinal by beta-carotene 15,15'-monoxygenase (BCMO1). However, beta-carotene absorption and conversion into retinal is extremely variable among individuals, with proportions of low responders to dietary beta-carotene as high as 45%. Recently, 2 common nonsynonymous single nucleotide polymorphisms (SNPs) within the BCMO1 coding region (R267S; rs12934922 and A379V; rs7501331) revealed reduced catalytic activity, confirming that genetic variations contribute to the low responder phenotype. Because 4 SNPs 5' upstream from the BCMO1 gene were recently shown to affect circulating carotenoid concentrations, the current study aimed to investigate the effects of these SNPs on beta-carotene conversion efficiency. Three of the 4 polymorphisms (rs6420424, rs11645428, and rs6564851) reduced the catalytic activity of BCMO1 in female volunteers by 59, 51, and 48%, respectively. The TG-rich lipoprotein fraction retinyl palmitate:beta-carotene ratio was negatively correlated with the G allele of rs11645428 (r = -0.44; P = 0.018), whereas it was positively correlated with the G allele of rs6420424 (r = 0.53; P = 0.004) and the T allele of rs6564851 (r = 0.41; P = 0.028). Furthermore, large inter-ethnic variations in frequency of affected alleles were detected, with frequencies varying from 43 to 84% (rs6420424), 52 to 100% (rs11645428), and 19 to 67% (rs6564851). In summary, a range of SNPs can influence the effectiveness of using plant-based provitamin A carotenoids to increase vitamin A status in at-risk population groups and this effect may vary depending on ethnic origin.

Beta-carotene conversion to vitamin A decreases as the dietary dose increases in humans

            (Novotny, Harrison et al. 2010) Download

It has been suggested that high doses of beta-carotene limit its conversion to vitamin A, yet this effect has not been well established in humans. A feeding study was conducted in a randomized crossover design in which volunteers consumed 2 doses of deuterium-labeled beta-carotene on 2 occasions, with beta-carotene and vitamin A response assessed by plasma area under the concentration time curve (AUC). Seven volunteers (4 men, 3 women) consumed each of 2 doses of beta-carotene-d8 and provided serial blood samples for 37 d after each dose. beta-Carotene doses were 20 and 40 mg. Plasma beta-carotene-d8 was assessed by HPLC-MS. Plasma retinol (ROH)-d4, which was derived from the beta-carotene-d8, was evaluated by GC-MS after saponification to convert retinyl esters to ROH prior to the formation of the trimethylsilylether. The plasma AUC for beta-carotene-d8 increased 2-fold from the 20-mg dose to the 40-mg dose. The plasma AUC for ROH-d4 increased 36% from the 20-mg dose to the 40-mg dose. These results establish that, in humans, beta-carotene conversion to vitamin A decreases as the dietary dose increases.

Bioconversion of dietary provitamin A carotenoids to vitamin A in humans

            (Tang 2010) Download

Recent progress in the measurement of the bioconversion of dietary provitamin A carotenoids to vitamin A is reviewed in this article. Methods to assess the bioavailability and bioconversion of provitamin A carotenoids have advanced significantly in the past 10 y, specifically through the use of stable isotope methodology, which includes the use of labeled plant foods. The effects of the food matrix on the bioconversion of provitamin A carotenoids to vitamin A, dietary fat effects, and the effect of genotype on the absorption and metabolism of beta-carotene have been reported recently. A summary of the major human studies that determined conversion factors for dietary beta-carotene to retinol is presented here, and these data show that the conversion efficiency of dietary beta-carotene to retinol is in the range of 3.6-28:1 by weight. There is a wide variation in conversion factors reported not only between different studies but also between individuals in a particular study. These findings show that the vitamin A value of individual plant foods rich in provitamin A carotenoids may vary significantly and need further investigation.

Retinoic acid attenuates beta-amyloid deposition and rescues memory deficits in an Alzheimer's disease transgenic mouse model

            (Ding, Qiao et al. 2008) Download

Recent studies have revealed that disruption of vitamin A signaling observed in Alzheimer's disease (AD) leads to beta-amyloid (Abeta) accumulation and memory deficits in rodents. The aim of the present study was to evaluate the therapeutic effect of all-trans retinoic acid (ATRA), an active metabolite of vitamin A, on the neuropathology and deficits of spatial learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, a well established AD mouse model. Here we report a robust decrease in brain Abeta deposition and tau phosphorylation in the blinded study of APP/PS1 transgenic mice treated intraperitoneally for 8 weeks with ATRA (20 mg/kg, three times weekly, initiated when the mice were 5 months old). This was accompanied by a significant decrease in the APP phosphorylation and processing. The activity of cyclin-dependent kinase 5, a major kinase involved in both APP and tau phosphorylation, was markedly downregulated by ATRA treatment. The ATRA-treated APP/PS1 mice showed decreased activation of microglia and astrocytes, attenuated neuronal degeneration, and improved spatial learning and memory compared with the vehicle-treated APP/PS1 mice. These results support ATRA as an effective therapeutic agent for the prevention and treatment of AD.

Vitamin A as a regulator of antigen presenting cells

            (Duriancik, Lackey et al. 2010) Download

Vitamin A has been long associated with immune system competence. Vitamin A deficiency is known to compromise many aspects of both innate and adaptive immune responses. Recent advances in retinol uptake and metabolism have identified the antigen presenting cell (APC) as a central immune cell capable of vitamin A metabolism. APC are now known to express retinaldehyde dehydrogenase and secrete retinoic acid. The retinoic acid produced has both autocrine and paracrine effects. Autocrine effects include upregulation of CD1d nonclassical major histocompatibility class I-like molecule and matrix metalloproteinase-9. Paracrine effects influence multiple lymphocyte lineage cell populations. Specifically, retinoic acid increases IgA isotype class switching by B lymphocytes, enhances regulatory T cell differentiation, and directs homing of lymphocytes to mucosa. CD1d lipid antigen presentation expands natural killer T cell populations. Previously, the focus of vitamin A action in adaptive immunity was on lymphocytes, but these recent advances suggest the APC may be the central player in carrying out the immune system functions of vitamin A.

An uncommon cause of esophagitis. Answer to the clinical challenges and images in GI question: image 1: esophageal hyperkeratosis secondary to vitamin A deficiency

            (Herring, Nowicki et al. 2010) Download

Vitamin A supplementation for preventing morbidity and mortality in children from 6 months to 5 years of age

            (Imdad, Herzer et al. 2010) Download

BACKGROUND: Vitamin A deficiency (VAD) is a major public health problem in low and middle income countries affecting 190 million children under 5. VAD can lead to many adverse health consequences, including death. OBJECTIVES: To evaluate the effect of vitamin A supplementation (VAS) for preventing morbidity and mortality in children aged 6 months to 5 years. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL 2010 Issue 2), MEDLINE (1950 to April Week 2 2010), EMBASE (1980 to 2010 Week 16), Global Health (1973 to March 2010), Latin American and Caribbean Health Sciences (LILACS), metaRegister of Controlled Trials and African Index Medicus (27 April 2010). SELECTION CRITERIA: Randomised controlled trials (RCTs) and cluster RCTs evaluating the effect of synthetic VAS in children aged 6 months to 5 years living in the community. We excluded studies concerned with children in hospital and children with disease or infection. We excluded studies evaluating the effects of food fortification, consumption of vitamin A rich foods or beta-carotene supplementation. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed studies for inclusion. Data were double abstracted and discrepancies were resolved by discussion. Meta-analyses were performed for outcomes including all-cause and cause-specific mortality, disease, vision, and side-effects. MAIN RESULTS: 43 trials involving 215,633 children were included. A meta-analysis for all-cause mortality included 17 trials comprising 194,795 children with 3536 deaths in both groups. At follow-up, there was a 24% observed reduction in the risk of all-cause mortality for Vitamin A compared with Control (Relative risk (RR) = 0.76 [95% confidence interval (CI) 0.69, 0.83]). Seven trials reported diarrhoea mortality and a 28% overall reduction for VAS (RR = 0.72 [0.57, 0.91]). There was no significant effect of VAS on cause specific mortality of measles, respiratory disease and meningitis. VAS reduced incidence of diarrhoea (RR = 0.85 [0.82, 0.87]) and measles morbidity (RR = 0.50 [0.37, 0.67]); however, there was no significant effect on incidence of respiratory disease or hospitalisations due to diarrhoea or pneumonia. There was an increased risk of vomiting within the first 48 hours of VAS (RR = 2.75 [1.81, 4.19]). AUTHORS' CONCLUSIONS: VAS is effective in reducing all-cause mortality by about 24% compared to no treatment. In our opinion, given the evidence that VAS causes considerable reduction in child mortality, further placebo-controlled trials of VAS in children between 6 months and 5 years of age are not required. There is a need for further studies comparing different doses and delivery mechanisms (for example, fortification).

Vitamin A antagonizes calcium response to vitamin D in man

         (Johansson and Melhus 2001) Download

For unknown reasons, the highest incidence of osteoporosis is found in northern Europe. In these populations, the sunlight exposure is limited and the vitamin A intake is high. The interaction between vitamin A and D has been the subject of several in vitro and animal studies. We have studied the acute effects of vitamin A and D on calcium homeostasis in 9 healthy human subjects. We compared the effect of (i) 15 mg of retinyl palmitate, (ii) 2 microg of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], (iii) 15 mg of retinyl palmitate plus 2 microg of 1,25(OH)2D3, and (iv) placebo in a double-blind crossover study. The subjects took vitamin preparations at 10:00 p.m. and the following day blood samples were collected five times from 8:00 a.m. to 4:00 p.m. Serum levels of 1,25(OH)2D3 and retinyl esters increased (1.7-fold and 8.3-fold, respectively; p < 0.01). As expected, serum calcium (S-calcium) increased (2.3%; p < 0.01) and S-parathyroid hormone (PTH) decreased (-32%; p < 0.05) after 1,25(OH)2D3 intake. In contrast, retinyl palmitate intake resulted in a significant decrease in S-calcium when taken alone (-1.0%; p < 0.05) and diminished the calcium response to 1,25(OH)2D3 after the combined intake (1.4%; p < 0.01). S-PTH was unaffected by retinyl palmitate. No significant changes in serum levels of the degradation product of C-telopeptide of type I collagen (CrossLaps), or U-calcium/creatinine levels were found. In conclusion, an intake of vitamin A corresponding to about one serving of liver antagonizes the rapid intestinal calcium response to physiological levels of vitamin D in man.

Developmental roles of the retinoic acid receptors

         (Lohnes, Mark et al. 1995) Download

Retinoic acid, one of the principle active metabolites of vitamin A (retinol), is believed to be essential for numerous developmental and physiological processes. Vitamin A deprivation (VAD) during development leads to numerous congenital defects. Previous studies of retinoic acid receptor (RAR) deficient mice failed to reveal any of these VAD-induced defects. This finding suggested that either the RARs are functionally redundant or that they are not critically required during development. In order to address these possibilities, we derived a number of RAR compound mutants. Unlike RAR single mutants, these compound null mutants died either in utero or shortly following birth. Histological analysis revealed essentially all of the defects characteristic of fetal VAD. A number of additional malformations, not described in previous VAD studies, were also observed. These included defects of the ocular and salivary glands and their ducts, the skeletal elements of the fore- and hindlimbs, and the cervical region of the axial skeleton. In addition, with the exception of derivatives forming within the first pharyngeal arch, most of the elements derived from mesectoderm emanating from cranial and hindbrain levels were affected. A number of these mutants also exhibited supernumerary cranial skeletal elements characteristics of the reptilian skull. A summary of the defects found in these RAR double mutants is presented.

The role of retinoic acid in embryonic and post-embryonic development

         (Maden 2000) Download

Retinoic acid (RA) is the bioactive metabolite of vitamin A (retinol) which acts on cells to establish or change the pattern of gene activity. Retinol is converted to RA by the action of two types of enzyme, retinol dehydrogenases and retinal dehydrogenases. In the nucleus RA acts as a ligand to activate two families of transcription factors, the RA receptors (RAR) and the retinoid X receptors (RXR) which heterodimerize and bind to the upstream sequences of RA-responsive genes. Thus, in addition to the well-established experimental paradigm of depriving animals of vitamin A to determine the role of RA in embryonic and post-embryonic development, molecular biology has provided us with two additional methodologies: knockout the enzymes or the RAR and RXR in the mouse embryo. The distribution of the enzymes and receptors, and recent experiments to determine the endogenous distribution of RA in the embryo are described here, as well as the effects on the embryo of knocking out the enzymes and receptors. In addition, recent studies using the classical vitamin A-deprivation technique are described, as they have provided novel insights into the regions of the embryo which crucially require RA, and the gene pathways involved in their development. Finally, the post-embryonic or regenerating systems in which RA plays a part are described, i.e. the regenerating limb, lung regeneration, hair cell regeneration in the ear and spinal cord regeneration in the adult.

Function of retinoic acid receptors during embryonic development

         (Mark, Ghyselinck et al. 2009) Download

Retinoids, the active metabolites of vitamin A, regulate complex gene networks involved in vertebrate morphogenesis, growth, cellular differentiation and homeostasis. Studies performed in vitro, using either acellular systems or transfected cells, have shown that retinoid actions are mediated through heterodimers between the RAR and RXR nuclear receptors. However, in vitro studies indicate what is possible, but not necessarily what is actually occurring in vivo, because they are performed under non-physiological conditions. Therefore, genetic approaches in the animal have been be used to determine the physiological functions of retinoid receptors. Homologous recombination in embryonic stem cells has been used to generate germline null mutations of the RAR- and RXR-coding genes in the mouse. As reviewed here, the generation of such germline mutations, combined with pharmacological approaches to block the RA signalling pathway, has provided genetic evidence that RAR/RXR heterodimers are indeed the functional units transducing the RA signal during prenatal development. However, due to (i) the complexity in "hormonal" signalling through transduction by the multiple RARs and RXRs, (ii) the functional redundancies (possibly artefactually generated by the mutations) within receptor isotypes belonging to a given family, and (iii) in utero or early postnatal lethality of certain germline null mutations, these genetic studies have failed to reveal all the physiological functions of RARs and RXRs, notably in adults. Spatio-temporally-controlled somatic mutations generated in given cell types/tissues and at chosen times during postnatal life, will be required to reveal all the functions of RAR and RXR throughout the lifetime of the mouse.


Vitamin A as a hormone: recent advances in understanding the actions of retinol, retinoic acid, and beta carotene

            (Ross and Ternus 1993) Download

Within the past few years, much has been learned about the metabolism and actions of vitamin A and the carotenoids. This article reviews the biochemical and cellular events in retinoid metabolism that lead to production of retinoic acid, an active metabolite of vitamin A. Retinoic acid functions in a hormone-like manner to regulate the expression of a number of genes. Beta carotene is now under study as an anticancer agent and for its possible beneficial effects in a number of chronic diseases. Current recommendations for carotene intake exceed the usual daily intake nearly fourfold.

Retinaldehyde is a substrate for human aldo-keto reductases of the 1C subfamily

            (Ruiz, Porte et al. 2011) Download

Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 muM). The kcat values were also low (0.18-0.6 min-1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.


Towards retinoid therapy for Alzheimer's disease

            (Shudo, Fukasawa et al. 2009) Download

Alzheimer's disease(AD) is associated with a variety of pathophysiological features, including amyloid plaques, inflammation, immunological changes, cell death and regeneration processes, altered neurotransmission, and age-related changes. Retinoic acid receptors (RARs) and retinoids are relevant to all of these. Here we review the pathology, pharmacology, and biochemistry of AD in relation to RARs and retinoids, and we suggest that retinoids are candidate drugs for treatment of AD.

Endogenous retinoids in mammalian growth plate cartilage: analysis and roles in matrix homeostasis and turnover

            (Williams, Kane et al. 2010) Download

The growth plate contains resting and proliferating chondrocytes in its upper zones (UGP) and maturing and hypertrophic chondrocytes in its lower zones (LGP), but the mechanisms by which it operates to sustain skeletal growth are not fully clear. Retinoid signaling was previously found to be nearly absent in UGP, but to be much stronger in LGP coincident with hypertrophy, extracellular matrix turnover and endochondral bone formation. To determine whether such distinct signaling levels and phenotypic events reflect different endogenous retinoid levels, the upper two-thirds and lower one-third of rabbit rib growth plates were microsurgically isolated and processed for ultrasensitive retinoid LC-tandem MS quantification. Indeed, the UGP samples contained only about a 0.6 nm concentration of all-trans-retinoic acid (atRA) that is the most active natural retinoid in tissues, whereas LGP samples contained nearly 3-fold higher atRA levels (about 1.8 nM). Perichondrium was quite rich in atRA (about 4.9 nM). Interestingly, the levels of retinol, the major but inactive atRA precursor, were similar in all tissues (1.1-1.6 muM), suggesting that the distinct atRA levels in UGP and LGP reflect different retinoid anabolic capacity. Indeed, RALDH2 and CRABP1 transcript levels were much higher in LGP than UGP samples. To determine the minimum effective atRA concentration, chondrogenic cells transfected with a retinoic acid response element (RARE)-luc reporter plasmid were treated with different concentrations of exogenous atRA (0-100 nM). About 3 nm atRA was needed to elicit appreciable RARE-luc reporter activity and to decrease proteoglycan synthesis and activity of an aggrecan enhancer reporter plasmid. In sum, the data indicate that (i) the endogenous levels of atRA are significantly higher in hypertrophic than upper zones of growth plate; (ii) such difference likely reflects distinct retinoid anabolic capacity; and (iii) importantly, atRA levels in hypertrophic portion are within effective ranges to elicit retinoid signaling and action, but those in upper zones are not.


References

Ding, Y., A. Qiao, et al. (2008). "Retinoic acid attenuates beta-amyloid deposition and rescues memory deficits in an Alzheimer's disease transgenic mouse model." J Neurosci 28(45): 11622-34.

Duriancik, D. M., D. E. Lackey, et al. (2010). "Vitamin A as a regulator of antigen presenting cells." J Nutr 140(8): 1395-9.

Grune, T., G. Lietz, et al. (2010). "Beta-carotene is an important vitamin A source for humans." J Nutr 140(12): 2268S-2285S.

Herring, W., M. J. Nowicki, et al. (2010). "An uncommon cause of esophagitis. Answer to the clinical challenges and images in GI question: image 1: esophageal hyperkeratosis secondary to vitamin A deficiency." Gastroenterology 139(2): e6-7.

Imdad, A., K. Herzer, et al. (2010). "Vitamin A supplementation for preventing morbidity and mortality in children from 6 months to 5 years of age." Cochrane Database Syst Rev(12): CD008524.

Johansson, S. and H. Melhus (2001). "Vitamin A antagonizes calcium response to vitamin D in man." J Bone Miner Res 16(10): 1899-905.

Lietz, G., J. Lange, et al. (2010). "Molecular and dietary regulation of beta,beta-carotene 15,15'-monooxygenase 1 (BCMO1)." Arch Biochem Biophys 502(1): 8-16.

Lietz, G., A. Oxley, et al. (2012). "Single nucleotide polymorphisms upstream from the beta-carotene 15,15'-monoxygenase gene influence provitamin A conversion efficiency in female volunteers." J Nutr 142(1): 161S-5S.

Lohnes, D., M. Mark, et al. (1995). "Developmental roles of the retinoic acid receptors." J Steroid Biochem Mol Biol 53(1-6): 475-86.

Maden, M. (2000). "The role of retinoic acid in embryonic and post-embryonic development." Proc Nutr Soc 59(1): 65-73.

Mark, M., N. B. Ghyselinck, et al. (2009). "Function of retinoic acid receptors during embryonic development." Nucl Recept Signal 7: e002.

Novotny, J. A., D. J. Harrison, et al. (2010). "Beta-carotene conversion to vitamin A decreases as the dietary dose increases in humans." J Nutr 140(5): 915-8.

Ross, A. C. and M. E. Ternus (1993). "Vitamin A as a hormone: recent advances in understanding the actions of retinol, retinoic acid, and beta carotene." J Am Diet Assoc 93(11): 1285-90; quiz 1291-2.

Ruiz, F. X., S. Porte, et al. (2011). "Retinaldehyde is a substrate for human aldo-keto reductases of the 1C subfamily." Biochem J 440(3): 335-44.

Shudo, K., H. Fukasawa, et al. (2009). "Towards retinoid therapy for Alzheimer's disease." Curr Alzheimer Res 6(3): 302-11.

Tang, G. (2010). "Bioconversion of dietary provitamin A carotenoids to vitamin A in humans." Am J Clin Nutr 91(5): 1468S-1473S.

Williams, J. A., M. Kane, et al. (2010). "Endogenous retinoids in mammalian growth plate cartilage: analysis and roles in matrix homeostasis and turnover." J Biol Chem 285(47): 36674-81.