PrediCTC, liquid biopsy in precision oncology: a technology transfer experience in the Spanish health system

Purpose. Management of metastatic disease in oncology includes monitoring of therapy response principally by imaging techniques like CT scan. In addition to some limitations, the irruption of liquid biopsy and its application in personalized medicine has encouraged the development of more efficient technologies for prognosis and follow-up of patients in advanced disease. Methods. PrediCTC constitutes a panel of genes for the assessment of circulating tumor cells (CTC) in metastatic colorectal cancer patients, with demonstrated improved efficiency compared to CT scan for the evaluation of early therapy response in a multicenter prospective study. In this work, we designed and developed a technology transfer strategy to define the market opportunity for an eventual implementation of PrediCTC in the clinical practice. Results. This included the definition of the regulatory framework, the analysis of the regulatory roadmap needed for CE mark, a benchmarking study, the design of a product development strategy, a revision of intellectual property, a cost-effectiveness study and an expert panel consultation. Conclusion. The definition and analysis of an appropriate technology transfer strategy and the correct balance among regulatory, financial and technical determinants are critical for the transformation of a promising technology into a viable technology, and for the decision of implementing liquid biopsy in the monitoring of therapy response in advanced disease

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PrediCTC, liquid biopsy in precision oncology: a technology transfer experience in the Spanish health system.

PURPOSE: Management of metastatic disease in oncology includes monitoring of therapy response principally by imaging techniques like CT scan. In addition to some limitations, the irruption of liquid biopsy and its application in personalized medicine has encouraged the development of more efficient technologies for prognosis and follow-up of patients in advanced disease. METHODS: PrediCTC constitutes a panel of genes for the assessment of circulating tumor cells (CTC) in metastatic colorectal cancer patients, with demonstrated improved efficiency compared to CT scan for the evaluation of early therapy response in a multicenter prospective study. In this work, we designed and developed a technology transfer strategy to define the market opportunity for an eventual implementation of PrediCTC in the clinical practice. RESULTS: This included the definition of the regulatory framework, the analysis of the regulatory roadmap needed for CE mark, a benchmarking study, the design of a product development strategy, a revision of intellectual property, a cost-effectiveness study and an expert panel consultation. CONCLUSION: The definition and analysis of an appropriate technology transfer strategy and the correct balance among regulatory, financial and technical determinants are critical for the transformation of a promising technology into a viable technology, and for the decision of implementing liquid biopsy in the monitoring of therapy response in advanced disease.

Conjugation of SUMO to p85 leads to a novel mechanism of PI3K regulation.

Class IA phosphatidylinositol 3-kinases (PI3Ks) are composed of p110 catalytic and p85 regulatory subunits. How regulatory subunits modulate PI3K activity remains only partially understood. Here we identified SUMO (small ubiquitin-related modifier) as a new player modulating this regulation. We demonstrate that both p85ß and p85α are conjugated to SUMO1 and SUMO2. We identified two lysine residues located at the inter-SH2 domain on p85ß, a critical region required for inhibition of p110, as being required for SUMO conjugation. A SUMOylation-defective mutant p85ß shows higher activation of the PI3K pathway, and increased cell migration and transformation. Moreover, the cancer-related KS459del mutant in p85α was less efficiently SUMOylated compared with the wild-type protein. Finally, our results show that SUMO modulates p85 tyrosine phosphorylation, a modification correlating with PI3K pathway activation. Thus, SUMO reduces the levels of tyrosine-phosphorylated-p85 while loss of SUMOylation results in increased tyrosine phosphorylation of p85. In summary, we identify SUMO as a new important player in the regulation of the PI3K pathway through modulation of p85.

Endometrial carcinoma: molecular alterations involved in tumor development and progression.

In the western world, endometrial carcinoma (EC) is the most common cancer of the female genital tract. The annual incidence has been estimated at 10-20 per 100,000 women. Two clinicopathological variants are recognized: the estrogen related (type I, endometrioid) and the non-estrogen related (type II, non-endometrioid).The clinicopathological differences are paralleled by specific genetic alterations, with type I showing microsatellite instability and mutations in phosphatase and tensin homologue deleted on chromosome 10, PIK3CA, K-RAS and CTNNB1 (ß-catenin), and type II exhibiting TP53 mutations and chromosomal instability. Some non-endometrioid carcinomas probably arise from pre-existing endometrioid carcinomas as a result of tumor progression and, not surprisingly, some tumors exhibit combined or mixed features at the clinical, pathological and molecular levels. In EC, apoptosis resistance may have a role in tumor progression. Understanding pathogenesis at the molecular level is essential in identifying biomarkers for successful targeted therapies. In this review, the genetic changes of endometrial carcinogenesis are discussed in the light of the morphological features of the tumors and their precursors.

The EMT signaling pathways in endometrial carcinoma

Endometrial cancer (EC) is the most common gynecologic malignancy of the female genital tract and the fourth most common neoplasia in women. In EC, myometrial invasion is considered one of the most important prognostic factors. For this process to occur, epithelial tumor cells need to undergo an epithelial to mesenchymal transition (EMT), either transiently or stably, and to differing degrees. This process has been extensively described in other types of cancer but has been poorly studied in EC. In this review, several features of EMT and the main molecular pathways responsible for triggering this process are investigated in relation to EC. The most common hallmarks of EMT have been found in EC, either at the level of E-cadherin loss or at the induction of its repressors, as well as other molecular alterations consistent with the mesenchymal phenotype-like L1CAM and BMI-1 up-regulation. Pathways including progesterone receptor, TGFβ, ETV5 and microRNAs are deeply related to the EMT process in EC (AU)

Tumor invasion and oxidative stress: biomarkers and therapeutic strategies.

Tumor invasion is paradigmatic of the complex interactions connecting a carcinoma with its environment, and a reflex of the cellular and molecular heterogeneity that defines the initiation of dissemination and metastasis. The hostile situation generated by a growing carcinoma and a reactive stroma is at the basis of the promotion of carcinoma invasion and metastasis, with oxidative stress emerging as a main player in the acquisition of an aggressive tumor phenotype. In this review, we present this complex scenario with a focus on the contribution of the reactive environment and the oxidative stress to the cellular and molecular events associated with carcinoma invasion and metastasis. We also discuss the potential of oxidative stress as a source of biomarkers of advance disease, and as supplier of a therapeutic armamentarium against the initial steps of metastatic dissemination.

ETV5 cooperates with LPP as a sensor of extracellular signals and promotes EMT in endometrial carcinomas.

Endometrial carcinoma (EC) is the most frequent among infiltrating tumors of the female genital tract, with myometrial invasion representing an increase in the rate of recurrences and a decrease in survival. We have previously described ETV5 transcription factor associated with myometrial infiltration in human ECs. In this work, we further investigated ETV5 orchestrating downstream effects to confer the tumor the invasive capabilities needed to disseminate in the early stages of EC dissemination. Molecular profiling evidenced ETV5 having a direct role on epithelial-to-mesenchymal transition (EMT). In particular, ETV5 modulated Zeb1 expression and E-Cadherin repression leading to a complete reorganization of cell-cell and cell-substrate contacts. ETV5-promoted EMT resulted in the acquisition of migratory and invasive capabilities in endometrial cell lines. Furthermore, we identified the lipoma-preferred partner protein as a regulatory partner of ETV5, acting as a sensor for extracellular signals promoting tumor invasion. All together, we propose ETV5-transcriptional regulation of the EMT process through a crosstalk with the tumor surrounding microenvironment, as a principal event initiating EC invasion.

Bioavailability of zinc from NutriSet zinc tablets compared with aqueous zinc sulfate.

BACKGROUND/OBJECTIVES: The apparent widespread extent of zinc (Zn) deficiency in developing countries and the efficacy of oral Zn supplements as an adjunct to oral rehydration therapy make oral Zn supplementation an increasingly important modality in clinical medicine and public health. In this study we aimed to compare the relative bioavailability of oral doses of 30 mg of Zn in two dosing forms. SUBJECTS/METHODS: In total, 10 healthy male volunteers ingested oral Zn doses with 200 ml plain water at about 0830 hours in the fasting state on two occasions, once as 30 mg of Zn in an aqueous solution of reagent grade zinc sulfate (ZnSO(4)) and another time as 1.5 NutriSet Zn tablets (Nutriset, Malaunay, France); on a third occasion, only plain water was consumed. Venous blood specimens were collected at baseline, 60, 120, 180 and 240 min after ingestion and the plasma Zn was measured for each sample. RESULTS: The relative bioavailability of oral Zn from a commonly used, tableted (NutriSet) form is only about half of that of a reference dose of aqueous ZnSO(4) as indicated by the area under the curve of serial plasma Zn excursion and maximal change in circulating Zn. CONCLUSIONS: Reduced or absent functional outcomes in Zn intervention trials may derive, in part, from a lower than anticipated intestinal uptake of the Zn in the tableted form.

A transcriptional signature associated with the onset of benign prostate hyperplasia in a canine model.

BACKGROUND: Benign prostatic hyperplasia (BPH) represents the most frequent proliferative abnormality of the human prostate. In spite of the well-characterized architectural development of BPH, little is known about the cellular and molecular events that contribute to it. METHODS: We have developed an animal model to evaluate the follow-up of hormone-induced BPH and the analysis of the gene expression associated with BPH. Immunohistochemistry on human patient samples validated the BPH-related molecular alterations. RESULTS: Canine specific Affymetrix microarray analysis performed on sequential biopsies obtained from a beagle dog dynamic model characterized a number of genes altered during the onset of BPH. In addition to the genes involved in calcification, matrix remodeling, detoxification, cell movement, and mucosa protection (MGP, MMP2, TIMP2, ITIH3, GST, MT2A, SULT1A1, FKBP1B, MUC1, STRBP, TFF3), the up-regulation of TGFB3 and CLU indicated a complete adjustment of the transdifferentiation, senescence and apoptosis programs. The up-regulation of Clusterin was validated by RT-qPCR and immunohistochemistry, both in the dog dynamic model and in human samples, further confirming the suitability of the animal model for the study of the molecular alterations associated with BPH. CONCLUSIONS: Transcriptome analysis performed on a dynamic animal model that accurately mimicked the human clinic, allowed us to characterize a gene expression pattern associated with the onset of BPH.

Novel molecular profiles of endometrial cancer-new light through old windows.

Endometrial carcinoma (EC) is the most common gynecological malignancy in the western world. A widely accepted dualistic model, which has been established on a morphological basis, differentiates EC into two broad categories: Type I oestrogen-dependent adenocarcinoma with an endometrioid morphology and Type II non-oestrogen-dependent EC with a serous papillary or clear cell morphology. Molecular genetic evidence indicates that endometrial carcinoma, as described in other malignancies, likely develops as the result of a stepwise accumulation of alterations in cellular regulatory pathways, such as oncogene activation and tumor suppressor gene inactivation, which lead to dysfunctional cell growth. These molecular alterations appear to be specific in Type I and Type II cancers. In type I endometrioid endometrial cancer, PTEN gene silencing in conjunction with defects in DNA mismatch repair genes, as evidenced by the microsatellite instability phenotype, or mutations in the K-ras and/or beta-catenin genes, are recognized major alterations, which define the progression of the normal endometrium to hyperplasia, to endometrial intraepithelial neoplasia, and then on to carcinoma. In contrast, Type II cancers show mutations of TP53 and Her-2/neu and seem to arise from a background of atrophic endometrium. Nevertheless, despite the great effort made to establish a molecularly-based histological classification, the following issues must still be clarified: what triggers the tumor cells to invade the myometrium and what causes vascular or lymphatic dissemination, finally culminating in metastasis? RUNX1, a transcription factor, was recently identified as one of the most highly over-expressed genes in a microarray study of invasive endometrial carcinoma. Another candidate gene, which may be associated with an initial switch to myometrial infiltration, is the transcription factor ETV5/ERM. These studies, as well as those conducted for other genes possibly involved in the mitotic checkpoint as a major mechanism of carcinogenesis in non-endometrioid endometrial cancer, could help in understanding the differences in the biology and the clinical outcome among histological types.

Molecular determinants of invasion in endometrial cancer.

Endometrial carcinoma is the most common gynaecological malignancy in the western world and the most frequent among infiltrating tumours of the female genital tract. Despite the characterisation of molecular events associated with the development of endometrial carcinoma, those associated with the early steps of infiltration and invasion in endometrial cancer are less known. Deep myometrial invasion correlates with more undifferentiated tumours, lymph-vascular invasion, node affectation and decreased global survival. In this review we present an overview of the molecular pathology of myometrial infiltration that defines the initial steps of invasion in endometrial cancer. Down-regulation of E-cadherin as a main player of epithelial to mesenchymal transition, as well as modifications on other molecules involved in cell-cell contacts, render cells with a migratory phenotype. In addition, altered signalling pathways and transcription factors associate with myometrial invasion, histologic grade and metastasis.

Molecular determinants of invasion in endometrial cancer

Endometrial carcinoma is the most common gynaecological malignancy in the western world and the most frequent among infiltrating tumours of the female genital tract. Despite the characterisation of molecular events associated with the development of endometrial carcinoma, those associated with the early steps of infiltration and invasion in endometrial cancer are less known. Deep myometrial invasion correlates with more undifferentiated tumours, lymph-vascular invasion, node affectation and decreased global survival. In this review we present an overview of the molecular pathology of myometrial infiltration that defines the initial steps of invasion in endometrial cancer. Down-regulation of E-cadherin as a main player of epithelial to mesenchymal transition, as well as modifications on other molecules involved in cell-cell contacts, render cells with a migratory phenotype. In addition, altered signalling pathways and transcription factors associate with myometrial invasion, histologic grade and metastasis (AU)

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Expression of androgen, oestrogen alpha and beta, and progesterone receptors in the canine prostate: differences between normal, inflamed, hyperplastic and neoplastic glands.

The expression of receptor for androgen (AR), oestrogen alpha and beta (ERalpha and ERbeta) and progesterone (PR) was examined immunohistochemically in canine prostate specimens (normal, hyperplastic, inflamed [prostatitis] or neoplastic). AR immunolabelling was seen in 100% of epithelial cells of normal and hyperplastic tissue, the corresponding figures for inflamed and carcinomatous tissue being 74% and 65%, respectively. ERalpha labelling was seen in 85% of epithelial cells in normal prostate glands, the corresponding figures for hyperplastic, inflamed and neoplastic glands being 35%, 22% and 12%, respectively. ERbeta labelling was seen in 85% of epithelial cells of normal glands and in about 70% of such cells in glands showing pathological changes. On the other hand, PR expression (weak) in normal glands was observed in fewer epithelial cells (44%) than in hyperplastic (70%), inflamed (62%) or neoplastic (64%) glands.

Molecular pathology of endometrial carcinoma: transcriptional signature in endometrioid tumors.

A dualistic model, which has been established on a morphological basis and that differentiates type I endometrioid from type II non-endometrioid endometrial cancer, is widely accepted. Molecular genetics have provided us with data supporting the dualistic model of endometrial tumorigenesis and with some clues to speculate about the sequence of the molecular alterations defining the tumorigenesis pathways. In type I endometrioid endometrial cancer, PTEN gene silencing, microsatellite instability associated with defects in DNA mismatch repair genes, or mutations in the K-ras gene are the known major alterations defining the progression from normal endometrium to hyperplasia and then on to carcinoma. Recently, cDNA microarray technology for identifying the differences in gene expression patterns between the histological types of endometrial cancer have permitted the identification of differentially expressed genes that could help us to understand differences in the biology and the clinical outcome between histiotypes. Genes involved in the mitotic checkpoint as a major mechanism of carcinogenesis in non-endometrioid endometrial cancer, or altered genes associated with the initial steps of myometrial infiltration in endometrioid endometrial cancer, represent examples of how useful large genetic screenings can be for understanding the tumorigenesis process and the future directions in the molecular pathogenesis of endometrial cancer.

Haematological response to haem iron or ferrous sulphate mixed with refried black beans in moderately anaemic Guatemalan pre-school children.

OBJECTIVE: Combating iron deficiency in toddlers with iron-fortified food has proved difficult in countries with phytate-rich diets. For this purpose, a new haem iron preparation was developed. The study compared changes in iron status after administration of refried beans with beans fortified with a haem iron preparation or ferrous sulphate (FeSO4). DESIGN: In a masked, stratified-randomised intervention trial, children received five 156-g cans of refried black beans per week for 10 consecutive weeks. The beans-only (control), FeSO4 and haem iron groups were offered a cumulative dose of 155 mg, 1625 mg and 1700 mg of iron from the bean intervention, respectively. Haemoglobin (Hb) and ferritin concentrations were determined at baseline and after 5 and 10 weeks. Compliance was examined weekly. SETTING: A low-income community in Guatemala City. SUBJECTS: One hundred and ten children aged 12-36 months with initial Hb values between 100 and 115 g l(-1). RESULTS: The cumulative intake of beans was approximately 80% of that offered, signifying an additional approximately 1300 mg of either haem or inorganic iron in the corresponding treatment groups over 10 weeks. Hb concentrations increased by the order of 7.3-11.4 g l(-1) during the intervention, but without significant differences across treatments. Average ferritin concentrations were unaffected by treatment assignment. However, post hoc analysis by subgroups of initial high ferritin and initial low ferritin found the Hb increments after 10 weeks in the haem iron group (13.1+/-7.7 g l(-1)) to be significantly greater than the respective increases (6.8+/-11.2 and 6.4+/-8.5 g l(-1)) in the inorganic iron and beans-only groups. CONCLUSIONS: Canned refried beans are a candidate vehicle for fortificant iron. Given the improved colour and organoleptic properties imparted by haem iron added to refried beans, its additional potential for benefiting the iron status of consumers with iron deficiency may recommend it over FeSO4.

Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action.

Microtubules are highly dynamic cellular polymers made of alphabeta-tubulin and associated proteins. They play a key role during mitosis, participating in the exact organization and function of the spindle, and are critical for assuring the integrity of the segregated DNA. Therefore, they represent one of the more effective targets in current cancer therapy. Paclitaxel (Taxol) is the prototype of the taxane family of antitumor drugs, and it was the first natural product shown to stabilize microtubules. This unique mechanism of action is in contrast to other microtubule poisons, such as Vinca alkaloids, colchicine, and cryptophycines, which inhibit tubulin polymerization. Taxanes block cell cycle progression through centrosomal impairment, induction of abnormal spindles and suppression of spindle microtubule dynamics. Triggering of apoptosis by aberrant mitosis or by subsequent multinucleated G1-like state related to mitotic slippage, depends on cell type and drug schedule. The development of fluorescent derivatives of paclitaxel led us to locate spindle pole microtubules and centrosomes as main sub-cellular targets of cytotoxic taxoids in living cells. In this review we discuss these findings in the context of a cell cycle-dependent response to taxanes, based on the cellular targets, and the status of the implicated cell cycle checkpoints. We also review those events that can influence this response, like the different signal transduction pathways activated/inactivated in relation to Bcl-2 phosphorylation and induction of apoptosis, and the controversial role of the p53 status on cell sensitivity to paclitaxel. Finally, cell cycle-dependent resistance, an emerging concept in combination sequential chemotherapy, is discussed on the basis of the cell cycle-dependent mechanisms of action of taxanes.

Phase II study of gemcitabine, 5-fluorouracil, and leucovorin in patients with pancreatic cancer.

The primary goal of this phase II study was to determine the efficacy of gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) plus 5-fluorouracil in patients with pancreatic cancer. Eligibility criteria included nonresectable locally advanced or metastatic pancreatic adenocarcinoma and measurable disease. Gemcitabine at 1,000 mg/m(2) and leucovorin at 20 mg/m(2) were administered intravenously 30 minutes before 5-fluorouracil 600 mg/m(2), weekly for 3 of every 4 weeks. Twenty nine patients were enrolled. The overall response rate was 21% (95% confidence interval: 8% to 40%), consisting of one complete response and five partial responses; 16 patients (55%) had stable disease. Median survival was 8.4 months (95% confidence interval: 2.6 to 14.2), and actuarial 1-year survival was 36%. Neutropenia (grade 3 only) was reported in 3.4% of patients, but was generally of short duration. No thrombocytopenia or evidence of cumulative myelosuppression was observed. The only significant nonhematologic events were grade 3 diarrhea and alopecia (both 3.4%). Gemcitabine plus 5-fluorouracil is active and well tolerated compared with results reported for each of these single agents. Thus, this combination justifies future comparative clinical trials. Semin Oncol 28 (suppl 10):44-49.

Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death.

Microtubules offer a very large local concentration of binding sites for cytotoxic taxoids or for hypothetical endogenous regulators. Several compounds from diverse sources stabilize microtubules and arrest cell division similarly to the antitumour drug Taxol. We have investigated the subcellular location of the Taxol binding sites, employing a fluorescent taxoid (FLUTAX) that reversibly interacts with the Taxol binding sites of microtubules and induces cellular effects similar to Taxol. The specific binding of FLUTAX to a fraction of the available cellular binding sites effectively inhibits division of cultured human tumour cells at G(2)/M, and triggers apoptotic death. The loci of reversible binding, directly imaged in intact U937 cells treated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole microtubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX consist of microtubule bundles and abnormal mitoses, including monopolar spindles and highly fluorescent multipolar spindles. The multiple asters and monopolar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma cells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). Depending on the FLUTAX treatment, OVCAR-3 cells died from abnormal mitosis or from a subsequent interphase with double chromatin content and lobulated nuclei (micronuclei), indicating impairment of centrosome separation. Fragmented centrosomes could be observed in FLUTAX-treated non-transformed 3T3.A31 cells, which developed micronuclei but were resistant to apoptosis. These results strongly suggest that centrosomal impairment by taxoid binding during interphase, in addition to the suppression of the kinetochore microtubule dynamics in the mitotic spindle, is a primary cause of cell cycle de-regulation and cell death.

Effects of discontinuing coffee intake on iron deficient Guatemalan toddlers' cognitive development and sleep.

Coffee is commonly given daily to toddlers in Guatemala. Possible negative effects of coffee ingestion on cognitive development and sleep patterns were assessed in 132 children 12-24 months of age who had received coffee for > 2 months and were iron deficient on at least one indicator. Children were stratified by initial hemoglobin (A= anemic, Hgb < 10.5 g/dl; NA = 'non-anemic', Hgb > or = 10.5 g/dl) and were randomly assigned to an experimental group (S = substitute consisting of sugar and coloring), and a control group (C = continuation of coffee) (42 C-NA; 53 S-NA; 18 C-A; and 19 S-A). Anemic children were provided Fe supplements for 2-3 months. Compliance was assessed every 2 weeks. After 5 months, testers masked to treatment group and anemia evaluated children with the Bayley Scales of Infant Development II in a central location. Scores were the Mental Development Index (MDI), the Psychomotor Development Index (PDI), and scales from the Behavior Rating Scale (BRS). The child's sleep in the previous 24 h was assessed with a set of standardized sleep questions to the care giver on the first visit and every 2 weeks thereafter. No significant effects of treatment on test scores or BRS ratings were found. In the 24 h period reported on at the final visit, children in the Substitute group slept more during the night and overall (night plus naps) than children in the Coffee group, a difference not found at the first visit. No differences were found in sleep difficulty or number of times waking at night. Women's reported coffee intake per day during pregnancy was associated with lower BRS ratings, even after controlling for SES and child age. The effects of postnatal coffee ingestion in Guatemala were seen for sleep duration, but not for cognitive development. Prenatal coffee ingestion was negatively associated with Behavior Rating Scales and should be investigated further.

Assessment of total body stores of vitamin A in Guatemalan elderly by the deuterated-retinol-dilution method.

BACKGROUND: Deuterated retinol dilution (DRD) gives quantitative estimates of total body stores of vitamin A. OBJECTIVES: In elderly people, we studied 1) the time when an oral dose of deuterated vitamin A equilibrates with body stores, 2) whether serum ratios of deuterated to nondeuterated retinol (D:H) at 3 or 6 d postdosing predicted body stores, and 3) the ability of DRD to detect changes in the size of the body vitamin A pool. DESIGN: A 10-mg oral dose of [2H4]retinyl acetate was administered to 60-81-y-old Guatemalans (n = 47); percentage enrichment of serum retinol with deuterated retinol was determined at 1-3 time points per subject at 3, 6, 7, 14, 20, 21, and 54 d. In subjects from whom blood was obtained at 3 and 21 d (n = 15) and at 6 and 20 d (n = 9), total body stores were calculated by using the formula of Furr et al (Am J Clin Nutr 1989;49:713-6) with 21- or 20-d data and correlated with serum D:H at 3 or 6 d postdosing. Nine subjects received diets containing 982+/-20 microg RE (x+/-SEM) plus 800 microg RE as retinyl acetate supplements for 32 d. DRD, serum retinol, and relative dose response were used to assess vitamin A status before and after the intervention. RESULTS: Deuterated retinol equilibrated with the body pool by 20 d postdosing. Vitamin A supplementation for 32 d increased body stores, although unexplained exaggerated increases were seen in some subjects. An inverse linear relation was found between estimates of body stores and serum D:H at 3 d postdosing (r = -0.75, P = 0.002); at 6 d postdosing, the correlation was weaker. CONCLUSIONS: DRD can detect changes in total body stores of vitamin A, although factors affecting serum D:H need to be elucidated. Serum D:H 3 d postdosing might be used as an early indicator of total body stores of vitamin A, although a predictive equation will need to be developed.