Biomedical subjects

M H Green

Publications and source records attributed to M H Green.

At least 19 recordsLinked to original sources

PubMed2000

UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome.

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.

Base Pairing↗

PubMed2000

The cancer-free phenotype in trichothiodystrophy is unrelated to its repair defect.

The DNA repair-deficient genetic disorders xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) can both result from mutations in the XPD gene, the sites of the mutations differing between the two disorders. The hallmarks of XP are multiple pigmentation changes in the skin and a greatly elevated frequency of skin cancers, characteristics that are not seen in TTD. XP-D and most TTD patients have reduced levels of DNA repair, but some recent reports have suggested that the repair deficiencies in TTD cells are milder than in XP-D cells. We reported recently that inhibition of intracellular adhesion molecule-1 (ICAM-1) expression by UVB irradiation was similar in normal and TTD cells but increased in XP-D cells, suggesting a correlation between ICAM-1 inhibition and cancer proneness. In the first part of the current work, we have extended these studies and found several other examples, including XP-G and Cockayne syndrome cells, in which increased ICAM-1 inhibition correlated with cancer proneness. However, we also discovered that a subset of TTD cells, in which arg112 in the NH2-terminal region of the XPD protein is mutated to histidine, had an ICAM-1 response similar to that of XP-D cells. In the second part of the work, we have shown that TTD cells with this specific NH2-terminal mutation are more sensitive to UV irradiation than other TTDs, most of which are mutated in the COOH-terminal region, and are indistinguishable from XP-D cells in cell killing, incision breaks, and repair of cyclobutane pyrimidine dimers. Because the clinical phenotypes of these patients do not obviously differ from those of TTDs with mutations at other sites, we conclude that the lack of skin abnormalities in TTD is independent of the defective cellular responses to UV. It is likely to result from a transcriptional defect, which prevents the skin abnormalities from being expressed.

Cell Line↗

PubMed2000

Kinetic analysis shows that iron deficiency decreases liver vitamin A mobilization in rats.

In view of evidence that nutritional status of iron and vitamin A may affect the other nutrient's metabolism, we used model-based compartmental analysis to examine effects of iron deficiency on whole-body vitamin A dynamics in rats. Weanling male Sprague-Dawley rats were fed the AIN93G diet with 2.5 nmol retinyl palmitate/g and either 45 [control (CN)] or 4 microg/g Fe [iron-deficient (ID)] for 8 wk. ID rats consumed food ad libitum; CN rats were food-restricted so that their body weights were the same as ID rats. Two rats/group were killed; liver vitamin A was determined and used for vitamin A balance calculations. [(3)H]Retinol-labeled plasma was administered intravenously to remaining rats, and 27 serial blood samples were collected for 7 wk. At killing, plasma vitamin A was 0.52+/-0.12 (ID, n = 5) vs. 1.34+/-0.12 micromol/L (CN, n = 6; P<0.001), and liver vitamin A was 809+/-94 (ID) vs. 112+/-24 nmol (CN, P<0.001). Plasma tracer data were fit to a three- or four-compartment model using the Simulation, Analysis and Modeling computer program and kinetic parameters were calculated. Vitamin A transfer rate between the retinyl ester storage pool [14+/-3 (ID) vs. 24+/-4 nmol/d (CN), P<0.05] and plasma was lower in ID rats. Vitamin A remained longer in the body [44+/-11 (ID) vs. 22+/-3 d (CN), P<0.05]. Adjusted mean disposal rate was lower in ID (10.0) than CN rats (19.9 nmol/d), as was estimated vitamin A absorption efficiency [58% (ID) vs. 76% (CN)]. Our results suggest that iron deficiency inhibits mobilization of vitamin A stores and may decrease the absorption and irreversible utilization of vitamin A.

Animals↗

PubMed2000

Mobilization of vitamin A stores in rats after administration of 2,3, 7,8-tetrachlorodibenzo-p-dioxin: a kinetic analysis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental contaminant that prevents the normal accumulation of vitamin A in liver and causes increased excretion of vitamin A. To determine what alterations in vitamin A metabolism occur first in response to TCDD treatment, we administered TCDD (7.0 microg/kg b.w. ) orally to rats that had received a nonperturbing (tracer) iv dose of [(3)H]vitamin A-labeled plasma (n = 3) or lymph (n = 3) 21 days earlier. Within a few days after TCDD administration, fraction of the injected radiolabel in plasma, which had been in a terminal slope when plotted on a semilog scale, increased and remained elevated until the experiment was terminated (day 42). At that time, liver vitamin A levels were 65% lower in TCDD-perturbed rats than in controls. Using model-based compartmental analysis and compartmental models developed previously for control rats (S. K. Kelley et al., 1998, Toxicol. Sci, 44:1-13), we determined the minimal changes needed to account for the perturbation in plasma [(3)H] tracer responses after TCDD administration. We determined that the effects of TCDD could be explained by adjusting the value of one fractional transfer coefficient corresponding to the mobilization of vitamin A from large, slowly turning-over pools. We speculate that this change corresponds to an increased fractional rate of retinyl ester hydrolysis, and that it precedes the TCDD-associated increased irreversible utilization and excretion of vitamin A.

Administration, Oral↗

PubMed2000

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes.

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.

Apoptosis↗

PubMed1999

The induction and activation of STAT1 by all-trans-retinoic acid are mediated by RAR beta signaling pathways in breast cancer cells.

Retinoic acid receptor-beta (RAR beta) and signal transducer and activator of transcription 1 (STAT1) are important mediators of the antiproliferative and apoptotic actions of retinoids and cytokines/growth factors, respectively. Expression of both RAR beta and STAT1 is lost in most breast cancer cell lines but it can be induced by retinoids in estrogen receptor-positive cells. We investigated a possible functional connection between these two mediators and present evidence supporting RAR beta as a tumor suppressor. First, by using different receptor-selective retinoids, we demonstrated that RAR beta induction in MCF-7 cells by all-trans-retinoic acid (atRA) was associated with the activation of STAT1 gene transcription. The direct involvement of RAR beta in atRA-induced STAT1 gene activation was further demonstrated by showing that transfection with an anti-sense RAR beta construct blocked atRA-induced STAT1 expression in MCF-7 cells whereas introduction of a sense-RAR beta construct resulted in STAT1 induction by atRA in MDA-MB 231 cells. In addition, we showed that STAT1 was phosphorylated/activated under atRA treatment of MCF-7 cells; this process required the involvement of RAR beta and protein synthesis. STAT1 phosphorylation/activation was accompanied by increased tyrosine kinase activity that was not due to the activation of JAK1, JAK2 or Tyk 2, suggesting the possible involvement of an unidentified tyrosine kinase.

Base Sequence↗

PubMed1999

Signal relay by retinoic acid receptors alpha and beta in the retinoic acid-induced expression of insulin-like growth factor-binding protein-3 in breast cancer cells.

Neither retinoic acid receptor-beta (RARbeta) nor insulin-like growth factor-binding protein-3 (IGFBP-3) is expressed in breast cancer cell line MCF-7. The expression of both proteins can be induced in response to all-trans-retinoic acid (atRA). By using an RARalpha-selective antagonist (Ro 41-5253), we demonstrated that RARbeta expression was induced by atRA through an RARalpha-dependent signaling pathway and that RARbeta induction was correlated with IGFBP-3 induction. However, MCF-7 cells transfected with sense RARbeta cDNA expressed IGFBP-3 even in the presence of the RARalpha-selective antagonist Ro 41-5253. On the other hand, antisense RARbeta cDNA transfection of MCF-7 cells blocked atRA-induced IGFBP-3 expression, indicating that RARbeta is directly involved in the mediation of IGFBP-3 induction by atRA. Induction of IGFBP-3 expression by atRA occurs at the transcriptional level, as measured by nuclear run-on assays. Finally, we showed that atRA-induced IGFBP-3 is functionally active in modulating the growth-promoting effect of IGF-I. These experiments indicate that RARalpha and RARbeta, both individually and together, are important in mammary gland homeostasis and breast cancer development. By linking IGFBP-3 to RARbeta, our experiments define the signal intersection between the retinoid and IGF systems in cell growth regulation and explain why loss of RARbeta might be critical in breast cancer carcinogenesis/progression.

Breast Neoplasms↗

PubMed1999

The 0.8% ultraviolet B content of an ultraviolet A sunlamp induces 75% of cyclobutane pyrimidine dimers in human keratinocytes in vitro.

Tanning lamps, emitting predominantly ultraviolet (UV) A, are used widely throughout the U.K. and other countries, but little is known about the long-term risks associated with their use, especially with respect to skin cancer. We have exposed normal human epidermal keratinocytes to a commercial tanning lamp and used the comet assay in association with DNA repair enzymes T4 endonuclease V and endonuclease III to investigate the relative yields of directly formed cyclobutane pyrimidine dimers (CPDs) and indirectly formed types of oxidative DNA damage. To put the risk of using tanning lamps into perspective, the sunbed used in this study (five Philips Performance 80W-R UVA tubes at a distance of 35 cm) was found to be approximately 0.7 times as potent at inducing CPDs as U.K. natural sunlight around noon on a fine summer day. This compares with a relative risk for CPD induction and erythema of 0.8 and 0.7 times, respectively, calculated from the relevant action spectra of tanning lamps and British noontime sunlight. To determine the relative contribution of UVB and UVA to the induction of CPDs and oxidative DNA damage, we modified the spectral output of the tanning lamps with a series of Schott WG UVB filters. The induction of CPDs was more dependent on the UVB component of the sunbed than oxidative types of damage. Schott WG UVB filters with 50% transmission at 305 nm reduced the yield of T4 endonuclease V sites by 42% while there was only a 17% decrease in the yield of endonuclease III sites. CPD induction was not completely abolished after irradiation through WG335 and WG345 nm filters despite there being no detectable UVB. From these data, it was estimated that, although the tanning lamps emitted only 0.8% of their total output in the UVB range, these wavelengths were responsible for the induction of over 75% of CPDs and 50% of the oxidative damage to DNA.

Cells, Cultured↗

PubMed1999

Frequencies of hprt(-) mutations and bcl-2 translocations in circulating human lymphocytes are correlated with United Kingdom sunlight records.

Between 1983 and 1995 we have monitored human populations for evidence of exposure to environmental mutagens, taking blood samples to measure hprt(-) mutant frequency in T cells and more recently bcl-2 t(14:18) translocation frequency in B cells. We have now analysed data from 785 assays on 448 blood samples from 308 normal subjects and find that there is a highly significant statistical correlation between hprt(-) mutant frequency and the sunlight record for the 3 weeks prior to taking the blood sample. We discuss the weaknesses in retrospective studies of this nature and the possibility of spurious epidemiological correlations that may result. More controlled experiments can be envisaged that would give a firmer basis to the statistical associations observed. hprt(-) mutations in T cells show little evidence of a UV fingerprint, so that the correlation may be due to immunomodulation rather than mutation. We also find a correlation between the sunlight record and bcl-2 translocation. This translocation is found at a low frequency in the B cells of many normal subjects and is the commonest translocation observed in non-Hodgkin's lymphoma. Our results strengthen the case for a link between sunlight and this increasingly common cancer.

Databases, Factual↗

PubMed1999

Possible effects of sunlight on human lymphocytes.

The human population is exposed to both the ultraviolet A (UVA) and B (UVB) regions of the solar spectrum. UVB induces mainly dipyrimidine photoproducts in DNA by a direct photochemical mechanism, whereas UVA is absorbed by other cellular constituents and induces mainly oxidative damage indirectly. The proportions of the different dipyrimidine photoproducts, and the ratio of dipyrimidine to oxidative damage depend on the exact spectral output of a UV source. Irradiation of human epidermal keratinocytes induces release of cytokines, with cyclobutane pyrimidine dimers playing a significant role in the process. These cytokines may then modulate the activity of cells of the immune system. Freshly isolated human lymphocytes are exquisitely sensitive to UVB irradiation, because of their low deoxyribonucleotide pools. They also have a separate defect in removal of cyclobutane pyrimidine dimers from their DNA. We have observed that frequencies of mutations at the hprt locus in human T-lymphocytes and translocations involving the bcl2 locus in B-lymphocytes appear to be associated with sunlight levels over the period before the blood sample was taken. This may be an indirect cytokine-mediated effect, and may be relevant to the possible link between non-Hodgkin's lymphoma and sunlight. On the other hand, sunlight can have beneficial effects, and may protect against autoimmune diseases including type I diabetes and multiple sclerosis.

Cell Transformation, Neoplastic↗

PubMed1999

Glucose kinetic responses to protein supplementation and exogenous somatotropin in late gestation dairy cows.

Glucose kinetics were measured in late gestation multiparous Holstein dairy cows fed diets with different amounts of dietary crude protein (13.3 vs. 17.8%), with and without exogenous somatotropin. The trial was conducted as a completely randomized design; 35 cows were used in the final analysis. Kinetic measurements were made using the single injection technique with uniformly labeled 13C-labeled glucose. A diet that contained 17.8% crude protein appeared to increase glucose utilization without a corresponding increase in supply. The evidence was an increased rate of glucose disposal. In contrast, exogenous bovine somatotropin (Posilac, Monsanto Co., St. Louis, MO) appeared to enhance glucose conservation, as indicated by a tendency for a decreased fractional catabolic rate and an increased glucose pool size. Somatotropin appeared to modify glucose metabolism in a pattern favorable for supporting terminal fetal development and lactogenesis and for maintaining or enhancing maternal glycemia. The latter pattern could have implications for improving the health of periparturient dairy cows.

Animals↗

PubMed1998

c-Myc is a major mediator of the synergistic growth inhibitory effects of retinoic acid and interferon in breast cancer cells.

The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-alpha were investigated. All-trans-retinoic acid and interferon-alpha acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-alpha by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-alpha down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc.Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-alpha, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-alpha. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-alpha: all-trans-retinoic acid + interferon-alpha --> upward arrow double-stranded RNA-dependent protein kinase --> downward arrow c-Myc --> cell growth inhibition.

Antineoplastic Agents↗

PubMed1998

Growth factor protection against cytokine-induced apoptosis in neonatal rat islets of Langerhans: role of Fas.

Treatment of neonatal rat islets of Langerhans with combined cytokines (interleukin-1beta 10(-10) M, tumour necrosis factor-alpha 10(-10) M, interferon-gamma 5 U/ml) led to extensive cell death, which was potentiated by Fas activation with the anti-Fas cytolytic antibody JO2. Pre-treatment with insulin (25 ng/ml) or insulin-like growth factor-1 (10(-8)M) gave only partial protection against cell killing, but prevented the Fas-mediated component. In the absence of cytokine treatment, Fas-mediated killing was not observed.

Animals↗

PubMed1998

Factors affecting the DNA damaging activity of superoxide and nitric oxide.

Nitric oxide and superoxide are formed endogenously and can react with each other and with other molecules to form a range of secondary and tertiary products. Some of these (e.g., peroxynitrite) are potent DNA-damaging agents and others (e.g., S-nitrosoglutathione) can act as reservoirs of the reactive species. Although the chemistry of these processes is now becoming understood, the question of which products are significant in vivo is not necessarily clear. To investigate these processes we have developed a cell-free version of the Comet assay, where the DNA from isolated nuclei is treated in agar on a microscope slide, following lysis. This offers an exceptionally sensitive assay for strand breakage in free DNA. Despite being present as a scavenger in the cell at millimolar levels, glutathione can act as a DNA-damaging pro-oxidant. Under appropriate conditions, glutathione-mediated damage is suppressed by superoxide dismutase and we suggest that superoxide may be a direct damaging agent, whose activity can be masked because of the involvement of superoxide in indirect mediation of damage or because of concomitant presence of hydroxyl radical.

Animals↗

PubMed1998

Use of the Comet assay to investigate the role of superoxide in glutathione-induced DNA damage.

Although glutathione is an important scavenging molecule within the cell, it can also act as a pro-oxidant and at biological concentrations (1 mM) can induce DNA damage. We have used a sensitive cell-free Comet assay for DNA strand breakage to investigate this damage and to try to determine the active species involved. We show a substantial protection against glutathione-mediated DNA damage by superoxide dismutase (200 U/ml) and complete protection by combined superoxide dismutase and catalase. Damage is also prevented by EDTA but only at 100 mM and is not prevented by the chelating agent diethylenetriamine-pentaacetic acid (100 microM). Although superoxide is known to potentiate DNA damage by other reactive species, none of these indirect mechanisms seem to account for our results and it is possible that superoxide may damage DNA directly. Under the same experimental conditions, S-nitrosoglutathione requires ultraviolet A photolysis to cause DNA strand breakage and superoxide dismutase increases the level of this damage. When intact human lymphocytes are incubated with glutathione (1 mM) in phosphate buffer, DNA damage is also observed, but in this case it is completely preventable by catalase, with no protective effect of superoxide dismutase. Since cellular scavenging systems are not completely protective against reactive species formed from autooxidation of extracellular glutathione and since glutathione and oxygen are ubiquitously present within cells, our results imply that cells may have a mechanism of preventing autooxidation, rather than simply relying on scavenging the reactive species formula.

Catalase↗

PubMed1998

Insulin secretion, DNA damage, and apoptosis in human and rat islets of Langerhans following exposure to nitric oxide, peroxynitrite, and cytokines.

Cytokine-induced damage may contribute to destruction of insulin-secreting beta-cells in islets of Langerhans during autoimmune diabetes. There is considerable controversy (i) whether human and rat islets respond differently to cytokines, (ii) the extent to which cytokine damage is mediated by induction of nitric oxide formation, and (iii) whether the effects of nitric oxide on islets can be distinguished from those of reactive oxygen species or peroxynitrite. We have analyzed rat and human islet responses in parallel, 48 h after exposure to the nitric oxide donor S-nitrosoglutathione, the mixed donor 3-morpholinosydnonimine, hypoxanthine/xanthine oxidase, peroxynitrite, and combined cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma). Insulin secretory response to glucose, insulin content, DNA strand breakage, and early-to-late stage apoptosis were recorded in each experiment. Rat islet insulin secretion was reduced by S-nitrosoglutathione or combined cytokines, but unexpectedly increased by peroxynitrite or hypoxanthine/xanthine oxidase. Effects on human islet insulin secretion were small; cytokines and S-nitrosoglutathione decreased insulin content. Both rat and human islets showed significant and similar levels of DNA damage following all treatments. Apoptosis in neonatal rat islets was increased by every treatment, but was at a low rate in adult rat or human islets and only achieved significance with cytokine treatment of human islets. All cytokine responses were blocked by an arginine analogue. We conclude: (i) Reactive oxygen species increased and nitric oxide decreased insulin secretory responsiveness in rat islets. (ii) Species differences lie mainly in responses to cytokines, applied at a lower dose and shorter time than in most studies of human islets. (iii) Cytokine effects were nitric oxide driven; neither reactive oxygen species nor peroxynitrite reproduced cytokine effects. (iv) Rat and human islets showed equal susceptibility to DNA damage. (v) Apoptosis was not the preferred death pathway in adult islets. (vi) We have found no evidence of human donor variation in the pattern of response to these treatments.

Adult↗

PubMed1998

Use of model-based compartmental analysis to study effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A kinetics in rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic, widespread environmental contaminant that has dramatic adverse effects on the metabolism of vitamin A. We used model-based compartmental analysis to investigate sites and quantitative impacts of TCDD on vitamin A kinetics in rats given on oral loading dose of TCDD in oil (3.5 micrograms/kg) followed by weekly maintenance doses (0.7 microgram/kg) or oil only. [3H]Retinol in its plasma transport complex (experiment 1) of lymph containing chylomicrons labeled mainly with [3H]retinyl esters (experiment 2) were administered i.v., and tracer kinetics in plasma, liver, carcass, urine, and feces were measured for up to 42 days. TCDD treatment caused significant reductions in liver vitamin A levels and significant changes in tracer kinetics and tracer excretion. A four-compartment model was used to fit tracer data for experiment 1; for experiment 2, compartments were added to describe the metabolism of newly absorbed vitamin A. The compartmental models predict that TCDD caused a slight delay in plasma clearance (via an increased recycling to plasma), and in liver processing, of chylomicron-derived vitamin A. Models for both experiments predict that TCDD exposure did not affect the fractional uptake of plasma retinol from the rapidly turning-over extravascular pool, but it doubled the fractional transfer of recycled retinol from slowly turning-over pools of vitamin A to plasma. The residence time for vitamin A was reduced by 70% in TCDD-treated rats, transfer into urine and feces was tripled, and vitamin A utilization rates were significantly increased. Since our results do not indicate that retinol esterification is inhibited, we hypothesize that some of the significant effects of TCDD on vitamin A metabolism result from increased catabolism and mobilization of vitamin A from slowly turning-over pools (especially the liver).

Animals↗

PubMed1998

Development of a compartmental model describing the dynamics of vitamin A metabolism in men.

Model-based compartmental analysis was used with the Simulation, Analysis and Modeling (SAAM) computer programs to analyze data on plasma retinoid kinetics in adult male subjects for 7 d after a single oral dose of 105 mumol of [8,9,19-13C]retinyl palmitate. We present here the data for one subject and discuss in detail the steps taken to develop a physiologically-based compartmental model that describes the dynamic behavior of plasma retinyl esters, [12C]retinol, [8,9,19-13C]retinol, and the sum of [12C] and [13C]retinol. First an absorption model was developed to fit data on the plasma appearance and disappearance of retinyl esters; this was used as input in development of models for labeled and unlabeled retinol. The large oral load of labeled vitamin A perturbed the unlabeled tracee system, and thus parallel models for tracer and tracee were developed; and a time-variant fractional transfer coefficient was incorporated into the tracee model. Following the absorption model, four-compartment models were developed to describe the dynamics of both labeled and unlabeled retinol. These models predict that, in spite of the large vitamin A load, the absorption efficiency was 34%; hepatic (presumably parenchymal cell) processing of the absorbed dose was essentially complete by 24 h; and, by 7 days, approximately 80% of the absorbed dose was in a compartment that presumably represents stored liver retinyl esters. The model also predicts that approximately 50 mumol of retinol passed through the plasma each day, compared to an estimated utilization rate of 4 mumol/day. This project provides unique and important information about whole-body vitamin A dynamic in humans, and presents approaches to specific modeling issues that may be encountered by others.

Adult↗