Acetyl zingerone: An efficacious multifunctional ingredient for continued protection against ongoing DNA damage in melanocytes after sun exposure ends.

OBJECTIVE: Recent research has shown that significant levels of cyclobutane pyrimidine dimers (CPDs) in DNA continue to form in melanocytes for several hours in the dark after exposure to ultraviolet radiation (UVR) ends. We document the utility of a new multifunctional ingredient, 3-(4-hydroxy, 3-methoxybenzyl)-pentane-2,4-dione (INCI acetyl zingerone (AZ)), to protect melanocytes against CPD formation after UVR exposure ends. METHODS: The use of AZ as an intervention to reduce CPD formation after irradiation was assessed in vitro by comparing kinetic profiles of CPD formation for several hours after irradiation in cells that were untreated or treated with AZ immediately after irradiation. Multifunctional performance of AZ as an antioxidant, quencher and scavenger was established using industry-standard in vitro chemical assays, and then, its efficacy in a more biological assay was confirmed by its in vitro ability to reduce intracellular levels of reactive oxygen species (ROS) in keratinocytes exposed to UVA radiation. Molecular photostability was assessed in solution during exposure to solar-simulated UVR and compared with the conventional antioxidant α-tocopherol. RESULTS: Even when added immediately after irradiation, AZ significantly inhibited ongoing formation of CPDs in melanocytes after exposure to UVA. Incubation with AZ before irradiation decreased intracellular levels of UVA-induced ROS formation in keratinocytes. Compared with α-tocopherol, the molecular structure of AZ endows it with significantly better photostability and efficacy to neutralize free radicals (∙OH, ∙OOH), physically quench singlet oxygen (1 O2 ) and scavenge peroxynitrite (ONOO- ). CONCLUSION: These results designate AZ as a new type of multifunctional ingredient with strong potential to extend photoprotection of traditional sunscreens and daily skincare products over the first few hours after sun exposure ends.

OBJECTIF: Une étude récente a montré que des taux significatifs de dimères cyclobutyliques de pyrimidine (Cyclobutane Pyrimidine Dimers, CPD) dans l'ADN continuaient à se former dans les mélanocytes pendant plusieurs heures, dans l'obscurité, après que leur exposition aux radiations ultraviolettes (UV) ait pris fin. Nous documentons l'utilité d'un nouvel ingrédient multifonctionnel, le 3-(4-hydroxy, 3- méthoxybenzyle)-pentane-2,4-dione (INCI acétyle zingérone (AZ)), pour protéger les mélanocytes contre la formation de CPD une fois l'exposition aux rayonnements UV terminée. MÉTHODES: L'utilisation d'AZ en tant qu'intervention pour réduire la formation de CPD après exposition aux ultraviolets a été évaluée in vitro en comparant les profils cinétiques de la formation de CPD pendant plusieurs heures après irradiation dans des cellules non traitées et dans des cellules traitées à l'AZ immédiatement après exposition. La performance multifonctionnelle de l'AZ comme agent antioxydant, absorbant et éliminateur a été établie à l'aide de dosages chimiques in vitro standard pour l'industrie, après quoi son efficacité à un dosage plus biologique a été confirmée par sa capacité in vitro à réduire les taux intracellulaires d'espèces réactives de l'oxygène (ROS) dans les kératinocytes exposés au rayonnement UV. La photostabilité moléculaire a été évaluée en solution pendant l'exposition UV simulée de rayonnements solaire et par rapport au traitement antioxydant conventionnel α-tocophérol. RÉSULTATS: Même lorsqu'il a été ajouté immédiatement après exposition, l'AZ a inhibé la formation continue de CPD dans les mélanocytes après l'exposition aux UV et ce de façon significative. Une incubation avec de l'AZ avant exposition a entraîné une diminution des taux intracellulaires de formation des ROS, induits par le rayonnement UV, dans les kératinocytes. Par rapport au α-tocophérol, la structure moléculaire de l'AZ lui confère une photostabilité significativement meilleure ainsi qu'une plus grande efficacité pour neutraliser les radicaux libres (∙OH, ∙OOH), absorber physiquement l'oxygène singulet (1 O2 ) et éliminer le peroxynitrite (ONOO- ). CONCLUSION: Ces résultats montrent que l'AZ, considéré comme un ingrédient multifonctionnel d'un type nouveau, jouit d'un fort potentiel de prolongation de l'effet photoprotecteur des écrans solaires traditionnels et des produits de soins de la peau pendant quelques heures après la fin de l'exposition au soleil.

The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas.

Basal cell carcinoma (BCC) is the most common cancer in humans. The majority of sporadic BCCs have allele loss on chromosome 9q22 implying that inactivation of a tumour suppressor in this region is an important step in BCC formation. The gene for nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominant disorder characterized by multiple BCCs, maps to the same region and is presumed to be the tumour suppressor inactivated at this site. NBCCS has been identified recently and encodes a protein with strong homology to the Drosophila segment polarity gene, patched. Analysis of Drosophila mutants indicates that patched interacts with the hedgehog signalling pathway, repressing the expression of various hedgehog target genes including wingless, decapentaplegic and patched itself. Using single strand conformational polymorphism (SSCP) to screen human patched in 37 sporadic BCCs, we detected mutations in one-third of the tumours. Direct sequencing of two BCCs without SSCP variants revealed mutations in those tumours as well suggesting that inactivation of patched is probably a necessary step in BCC development. Northern blots and RNA in situ hybridization showed that patched is expressed at high levels in tumour cells but not normal skin suggesting that mutational inactivation of the gene leads to overexpression of mutant transcript owing to failure of a negative feedback mechanism.

Sunlight and the onset of skin cancer.

The photons of sunlight precipitate a series of genetic events in skin leading to cancer. These events involve somatic mutations as well as inherited alleles. Competition between cell populations ensues, as a single mutated cell expands into a clone. Thus cancer involves both a single-cell problem and a many-cell problem; in skin cancer, sunlight appears to drive both.

Transgenic expression of survivin in keratinocytes counteracts UVB-induced apoptosis and cooperates with loss of p53.

The inhibitor of apoptosis protein survivin has been implicated in both cell cycle control and apoptosis resistance. To discriminate between these different roles, we used transgenic expression of survivin in the skin as a model for cell proliferation, differentiation, and apoptosis. Transgenic mice expressing survivin under the control of a keratin-14 promoter developed normally, without histologic abnormalities of the skin or hair, epidermal hyperplasia, or developmental abnormalities of basal or suprabasal epidermis. Keratinocyte proliferation assessed under basal conditions, or after ultraviolet-B (UVB) irradiation, or phorbol ester stimulation was unchanged in survivin transgenic mice. In contrast, survivin expression inhibited UVB-induced apoptosis in vitro and in vivo (i.e., sunburn cell formation), whereas it did not affect Fas-induced cell death. When crossed with p53 knockout mice, transgenic expression of survivin in a p53(+/-) background substituted for the loss of a second p53 allele and further inhibited UVB-induced apoptosis. These data provide the first in vivo evidence that survivin inhibits apoptosis and suggest that this pathway may oppose the elimination of cancerous cells by p53.

Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells.

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).

Relationship between sunlight exposure and a key genetic alteration in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common cancer in humans. Epidemiologic studies implicate sunlight exposure as one risk factor, but the limited association between BCCs and UVB radiation (i.e., UV radiation of a wavelength of 280-320 nm) suggests that additional factors must be involved. At the molecular level, not much is known about the role of specific environmental agents in the pathogenesis of BCCs. Point mutations of the types produced by UVB radiation are seen in the p53 gene (also known as TP53; chromosome 17p) of 40%-56% of BCCs. Loss of heterozygosity (LOH) on chromosome 9q22, however, is the most frequent genetic alteration in these tumors, and its causative agent is unknown. PURPOSE: We investigated whether the genetic alteration in chromosome 9 is common to all clinical subtypes of BCCs and whether inactivation of this putative tumor suppressor is related to sunlight exposure. The presence of UVB radiation-related point mutations in the p53 gene was used as an internal control for sunlight exposure to the precursor cells. METHODS: Tumor and blood samples were obtained from skin cancer patients by a surgeon who used Mohs' micrographic surgical technique. Clinical information on each tumor included location, size, histologic, subtype and whether it was primary or recurrent and sporadic or hereditary. Sixty BCCs from 58 patients were evaluated for LOH with 12 polymorphic markers that span chromosome 9. A subset of 18 tumors was evaluated for point mutations in exons 2-11 of the p53 gene, and a subset of 26 tumors was evaluated for LOH by use of a polymorphism in exon 4 of the p53 gene. Associations between tumor characteristics and molecular alterations were tested by a two-tailed chi-squared analysis or a two-tailed Fisher's exact test, depending on sample size. RESULTS: In a clinically diverse series of 47 informative tumors, 32 (68%) showed LOH for chromosome 9q markers, irrespective of histologic characteristics or clinical behavior. Forty-four (94%) of the 47 tumors were from sun-exposed areas of the body, defined as the head and neck in both sexes, shoulders or chest in males, and legs in females. No association was found between chromosome 9q LOH and sunlight exposure, as assessed by either the location of tumors on the body or the presence of UVB radiation-related p53 mutations. Of note, there was a striking difference between the frequency of LOH on chromosome 17p (two [12.5%] of 16 informative tumors) and on chromosome 9q (32 [68%] of 47 informative tumors; P < .001). CONCLUSIONS: Inactivation of a gene on chromosome 9q22 may be a necessary event for basal cell carcinogenesis. The pathogenesis of mutations in this gene may involve factors other than sunlight in a large proportion of tumors. IMPLICATIONS: The limited association between sunlight exposure and BCC incidence may reflect an etiologic contribution of additional environmental agents.

Antioxidant action via p53-mediated apoptosis.

The biological effects of antioxidants are often considered in terms of their effects on oxygen or lipid radicals. However, antioxidants can also exert their effects through altering the cellular redox potential. Herein, we report that sulfur-containing antioxidants such as N-acetylcysteine and dimercaptopropanol induced apoptosis in several transformed cell lines and transformed primary cultures but not in normal cells. In contrast, chain-breaking antioxidants such as vitamin E lacked this activity. An increased glutathione level was not required for apoptosis; however, all apoptosis-inducing antioxidants elevated the total cellular thiol levels. Antioxidant-induced apoptosis required the p53 tumor suppressor gene. N-Acetylcysteine elevated p53 expression posttranscriptionally by increasing the rate of p53 mRNA translation rather than by altering the protein stability. The p53 induction occurred in normal cells. These observations indicate a redox sensor for p53 induction in vivo, with additional transformation-specific information being required for apoptosis. Manipulating p53-dependent apoptosis with nontoxic antioxidants may have a direct clinical application.

Photosensitizing dyes for the selection of nontumorigenic revertants from human lung cancer cell lines.

Certain positively charged, lipophilic dyes have been noted by various authors to localize selectively in the mitochondria of carcinoma cells. Oseroff et al. (Proc. Natl. Acad. Sci. USA, 83:9729-9733, 1986) studied 10 carcinoma-specific mitochondrial photosensitizers and judged N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine (EDKC) to be the most effective in selective carcinoma cell photolysis, a system where light-absorbing molecules accumulate only in carcinoma cells and on illumination initiate a reaction that kills or damages those cells. The present study duplicated the published EDKC retention result for the normal monkey kidney epithelial cell line CV-1. A series of nontumorigenic and tumorigenic human bronchial epithelial and human pleural mesothelial cells were assayed for EDKC uptake and retention, with the intent of using selective carcinoma cell photolysis to isolate nontumorigenic revertants of the tumorigenic lung cell lines. In addition, the uptake and retention of the fluorescent, mitochondria- and carcinoma-specific dye rhodamine-123 were surveyed in a series of hybrids between tumorigenic and nontumorigenic human bronchial epithelial cells. The half-life of dye retention ranged from 6 to 12 h in all the bronchial epithelial and mesothelial cells studied, with little or no dye selectivity for tumorigenic cells. When EDKC-retaining bronchial epithelial cells were illuminated with red light, significant reductions in short term viability and colony-forming efficiency were seen, which became more pronounced as light and dye doses were increased. However, these effects did not correlate with tumorigenicity within the cell series. The method, therefore, does not appear generally useful for the selection of nontumorigenic variants of human bronchial epithelial or pleural mesothelial cancers of the lung.

Stimulatory effect of oral administration of green tea or caffeine on ultraviolet light-induced increases in epidermal wild-type p53, p21(WAF1/CIP1), and apoptotic sunburn cells in SKH-1 mice.

Pretreatment of SKH-1 mice with p.o.-administered 0.6% green tea (6 mg of lyophilized tea solids/ml) or 0.044% caffeine (0.44 mg/ml; concentration present in 0.6% green tea) for 2 weeks enhanced UV-induced increases in the number of p53-positive cells, p21(WAF1/CIP1)-positive cells, and apoptotic sunburn cells in the epidermis. These effects of p.o.-administered green tea or caffeine on early adaptive responses to UV provide the first demonstration of in vivo up-regulation of a tumor suppressor gene by a chemopreventive agent. The stimulatory effect of green tea and caffeine on UV-induced increases in the number of p53-positive cells, p21(WAF1/CIP1)-positive cells, and apoptotic sunburn cells may play a role in the inhibitory effects of tea and caffeine on UV-induced carcinogenesis.

Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes.

Neonatal human prostatic epithelial cells (NP-2s) were transfected by strontium phosphate coprecipitation with a plasmid (pRSV-T) containing the SV40 early region genes. The cells transfected with pRSV-T, but not the sham-transfected controls, formed rapidly growing, multilayered colonies within 2 weeks at a frequency of 1 x 10(-4) in a serum-free medium (P4-8F). In all, 28 colonies of transformed cells were isolated. Three of these have been cultured for a sufficient length of time to show that their growth potentials are well beyond that of the normal progenitor cells (NP-2s). There is also little or no indication of the culture "crisis" commonly seen in SV40-transformed cells in these transfected lines. All contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into nude mice. The transformed as well as the parental NP-2s cells require bovine pituitary extract for growth in serum-free medium and are stimulated by transforming growth factor beta 1 (TGF-beta 1) and epidermal growth factor in clonal growth assays. In contrast, a prostatic carcinoma cell line (PC-3) is inhibited by TGF-beta 1. This serum-free system and immortalized transfected clones will be useful for studying the action of putative prostatic carcinogens and tumor-promoting agents.

Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes.

Normal human bronchial epithelial cells were infected with SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium phosphate coprecipitation with plasmids containing the SV40 early region genes. Colonies of morphologically altered cells were isolated and cultured; these cells had extended culture lifespans compared to normal human bronchial epithelial cells. All cultures eventually underwent senescence, with the exception of one which appears to have unlimited proliferative potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonproducer cultures were analyzed further. The cells retained electron microscopic features of epithelial cells, and keratin and SV40 T-antigen were detected by indirect immunofluorescence. All of the cultures were aneuploid with karyotypic abnormalities characteristic of SV40-transformed cells. No tumors formed after s.c. injection of the cells in nude mice. These cells should be useful for studies of multistage bronchial epithelial carcinogenesis.

Intragenic domains of strand-specific repair in Escherichia coli.

Heterogeneity of DNA repair has been observed at different levels of genomic organization, including chromatin domains, expressed genes and DNA strands. If heterogeneity also existed intragenically, it could reveal fine details of the excision repair mechanism in vivo. Here we measure the frequency of UV-induced cyclobutane pyrimidine dimers at individual nucleotides within defined portions of two Escherichia coli genes, lacl and lacZ, at various times after irradiation. Two domains of differential repair rates were apparent, with repair being slow at nucleotides adjacent to the transcription start sites. In lacZ, the domain of faster repair began 32 bases downstream of the transcription start site and required the mfd gene. Since mfd codes for a transcription-repair coupling factor, this transcription-coupled repair system evidently becomes operative downstream of the initiation complex region in vivo. Unexpectedly, however, (1) an mfd mutation reduced repair in the downstream domain even when transcription was at a very low level and (2) induction of lacZ transcription with isopropyl-beta-D-thiogalactoside overcame this reduction. Evidently, the Mfd transcription-repair coupling factor is required for basal levels of strand-specific repair in this gene, but induced levels of repair are related to transcription through another mechanism.

The DNA damage signal for Mdm2 regulation, Trp53 induction, and sunburn cell formation in vivo originates from actively transcribed genes.

The stratum corneum and DNA repair do not completely protect keratinocytes from ultraviolet B. A third defense prevents cells with DNA photoproducts from becoming precancerous mutant cells: apoptosis of ultraviolet-damaged keratinocytes ("sunburn cells"). As signals for ultraviolet-induced apoptosis, some studies implicate DNA photoproducts in actively transcribed genes; other studies implicate non-nuclear signals. We traced and quantitated the in vivo DNA signal through several steps in the apoptosis-signaling pathway in haired mice. Homozygous inactivation of Xpa, Csb, or Xpc nucleotide excision repair genes directed the accumulation of DNA photoproducts to specific genome regions. Repair-defective Xpa-/- mice were 7-10-fold more sensitive to sunburn cell induction than wild-type mice, indicating that 86-90% of the ultraviolet B signal for keratinocyte apoptosis involved repairable photoproducts in DNA; the remainder involves unrepaired DNA lesions or nongenomic targets. Csb-/- mice, defective only in excising photoproducts from actively transcribed genes, were as sensitive as Xpa-/-, indicating that virtually all of the DNA signal originates from photoproducts in active genes. Conversely, Xpc-/- mice, defective in repairing the untranscribed majority of the genome, were as resistant to apoptosis as wild type. Sunburn cell formation requires the Trp53 tumor suppressor protein; 90-96% of the signal for its induction in vivo involved transcribed genes. Mdm2, which regulates the stability of Trp53 through degradation, was induced in vivo by low ultraviolet B doses but was suppressed at erythemal doses. DNA photoproducts in actively transcribed genes were involved in approximately 89% of the Mdm2 response.

Longevity-dependent organ-specific accumulation of DNA damage in two closely related murine species.

To measure directly the accumulation of DNA damage with age, and to understand better the effect of modulators of DNA damage in vivo, the DNA of brain, liver, and kidney of two mice from different families, Mus musculus and Peromyscus leucopus, have been examined for age-dependent accumulation of single-strand breaks plus alkali-labile bonds, by the alkaline sucrose sedimentation method. These two species of small rodents are closely related taxonomically, yet differ significantly in maximum achievable lifespan. Using the reciprocal of the number average molecular weight for estimation of DNA size, these analyses indicate that: (a) DNA damage does not measurably accumulate in brain tissue; (b) the accumulation of DNA damage was more pronounced in hepatic DNA than other tissue DNA; and (c) the rate of accumulation of DNA damage in liver and kidney cells with age was greater in the shorter-lived species (M. musculus) and was inversely proportional to maximum achievable lifespan. There are suggestions that a similar threshold might exist for tolerance of DNA damage in the two species in specific organs, and that these species differ in the rate at which this threshold is reached as a function of maximum achievable lifespan.

Local recurrence versus new primary: clinical analysis of 82 breast relapses and potential applications for genetic fingerprinting.

PURPOSE: The purpose of this study was to perform a detailed clinical pathological analysis of breast relapses in patients treated with conservative surgery and radiation therapy in an effort to classify those relapses as true local recurrences or second primary tumors, and to assess the prognostic and therapeutic implications of such a classification system. METHODS AND MATERIALS: Of 990 patients treated with conservative surgery and radiation therapy at our facilities prior to December 1987, 82 patients have experienced a relapse in the conservatively treated breast as the primary site of failure. Patients were classified as having new primary tumors if they fulfilled any one of the following criteria: a) breast relapse occurring at a site distinctly removed from the original tumor; b) histology of the breast relapse compared with the original tumor consistent with a new primary; or c) DNA flow cytometry converting from an aneuploid primary to a diploid relapse. RESULTS: As of 2/92, with a median follow-up of 5.4 years from the time of breast relapse, the overall 5-year survival rate following breast relapse was 55%. Forty-seven patients were classified as true recurrences and 33 patients were classified as new primaries. Patients classified as true recurrences had a shorter median time to breast relapse than patients classified as new primaries (3.16 years vs. 5.42 years, p < .05) and an inferior post breast recurrence survival rate compared to patients classified as new primaries (36% vs. 89%, p < .05). Residual disease outside of the recurrent tumor bed was also noted to be more frequent in patients classified as true recurrences compared to patients classified as new primaries (48% vs. 16%, p < .05). CONCLUSION: Based on the clinical and pathological criteria outlined, it appears that a significant portion of patients experiencing a relapse in the conservatively treated breast may have new primary tumors as opposed to true local relapses. Distinction between a true recurrence and a new primary tumor may have significant prognostic implications. Uncertainties associated with the clinical and pathological criteria are presented and further investigations with genetic fingerprinting techniques to establish the clonality of breast relapses are presented and discussed.

Status of the p53 tumor suppressor gene in human squamous carcinoma cell lines.

Dominant-negative and/or loss-of-function mutations of the p53 tumor suppressor gene are frequently found in squamous cell carcinomas of the skin and of the head-and-neck region. In order to identify the precise mechanisms of inactivation of p53 in tumors of this class, we examined the status of p53 RNA, protein and DNA in a panel of eight human squamous carcinoma cell lines (head-and-neck, 3; esophagus, 1; lung, 1; uterine cervix, 2; vulva, 1). Three lines (A253, CaLu-1, SqCC/Y1) failed to express any p53 mRNA. A253 cells contained a single p53 allele without mutations in exons 2-9, suggesting that the lack of transcription was the result of mutations in the regulatory region of the gene. Both p53 alleles were deleted in CaLu-1 cells, whereas the single allele present in SqCC/Y1 cells was rearranged and carried two missense mutations in exon 5. Two cell lines (A431, FaDu) expressed only 50% of the normal level of p53 mRNA, either because only one allele was present (A431), or because only one of the two alleles was transcribed (FaDu). The two cervical carcinoma lines (CaSki, C4-1) expressed normal levels of p53 mRNA, but no wild type protein, presumably as a result of accelerated degradation by the human papillomavirus 16 or -18 E6 oncoprotein present in these cells as previously described (Scheffner et al., Proc. Natl. Acad. Sci. USA 88:5523-5527; 1991). Three of the lines expressed only mutant p53 protein (A431, FaDu, CE-48) resulting from missense mutations in codons 248 and 273.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of single-strand breaks plus alkali-labile bonds by N-nitrosoureas in rat tissues in vivo: ethylnitrosourea versus benzylnitrosourea.

Alkaline sucrose sedimentation procedures were used to quantitate the amount of single-strand breaks plus alkali-labile bonds (SSB + ALB) induced and repaired following a single intraperitoneal injection of the neurocarcinogen N-ethyl-N-nitrosourea (ENU) and its non-neurocarcinogenic analog N-benzyl-N-nitrosourea (BNU) in the brain, liver and kidney of female Sprague-Dawley rats. SSB + ALB were measured and used as an indicator of apurinic/apyrimidinic sites, phosphotriesters and in situ breaks. ENU induced a dose-dependent increase in the number of SSB + ALB at the doses studied (0, 0.39, 0.77, 1.54 mmoles/kg) in all 3 tissues. At 1 h postinjection with 0.77 mmoles/kg of these compounds there were 50-70% fewer breaks induced by BNU than ENU. The SSB + ALB induced by ENU persisted over a 7-day period, while those induced by BNU did not. Thus, these studies showed that 2 homologues of nitrosoureas, ENU and BNU, exhibited different potentials to induce and to persist SSB + ALB in vivo.