1: J Clin Endocrinol Metab. 1988 Aug;67(2):373-8.
Influence of season and latitude on the cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin. Webb AR, Kline L, Holick MF. Vitamin D, Skin, and Bone Research Laboratory, Boston University Medical School, Massachusetts 02118.
Sunlight has long been recognized as a major provider of vitamin D for humans; radiation in the UVB (290-315 nm) portion of the solar spectrum photolyzes 7-dehydrocholesterol in the skin to previtamin D3, which, in turn, is converted by a thermal process to vitamin D3. Latitude and season affect both the quantity and quality of solar radiation reaching the earth's surface, especially in the UVB region of the spectrum, but little is known about how these influence the ability of sunlight to synthesize vitamin D3 in skin. A model has been developed to evaluate the effect of seasonal and latitudinal changes on the potential of sunlight to initiate cutaneous production of vitamin D3. Human skin or [3 alpha-3H]7-dehydrocholesterol exposed to sunlight on cloudless days in Boston (42.2 degrees N) from November through February produced no previtamin D3. In Edmonton (52 degrees N) this ineffective winter period extended from October through March. Further south (34 degrees N and 18 degrees N), sunlight effectively photoconverted 7-dehydrocholesterol to previtamin D3 in the middle of winter. These results quantify the dramatic influence of changes in solar UVB radiation on cutaneous vitamin D3 synthesis and indicate the latitudinal increase in the length of the "vitamin D winter" during which dietary supplementation of the vitamin may be advisable.
Research Support, U.S. Gov't, P.H.S. Seasons*
Skin/radiation effects Sunlight*
PMID: 2839537 [PubMed - indexed for MEDLINE]
Cancer Epidemiology Biomarkers & Prevention Vol. 14, 2303-2309, October 2005
1 Occupational Health Program, Department of Environmental Health, 2 Department of Biostatistics, Harvard School of Public Health, 3 Department of Nutrition, and 4 Department of Epidemiology, Harvard School of Public Health; 5 Department of Medicine and 6 Thoracic Surgery Unit, Department of Surgery, Massachusetts General Hospital, 7 Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, 8 Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Requests for reprints: David C. Christiani, Occupational Health Program, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. Phone: 617-432-1641; Fax: 617-432-6981. E-mail: firstname.lastname@example.org://www.pnas.org/cgi/reprint/92/8/3124.pdf
Recent Results Cancer Res. 2003;164:3-28.
Evolution and function of vitamin D.
Vitamin D Laboratory, Section of Endocrinology, Diabetes and Nutrition,
Department of Medicine, Boston University Medical Center, Boston, MA 02118, USA.
It is remarkable that phytoplankton and zooplankton have been producing vitamin
D for more than 500 million years. The role of vitamin D in lower non-vertebrate
life forms is not well understood. However, it is critically important that most
vertebrates obtain an adequate source of vitamin D, either from exposure to
sunlight or from their diet, in order to develop and maintain a healthy
mineralized skeleton. Vitamin D deficiency is an unrecognized epidemic in most
adults who are not exposed to adequate sunlight. This can precipitate and
exacerbate osteoporosis and cause the painful bone disease osteomalacia. Once
vitamin D is absorbed from the diet or made in the skin by the action of
sunlight, it is metabolized in the liver to 25-hydroxyvitamin D [25(OH)D] and
then in the kidney to 1,25-dihydroxyvitamin D [1,25(OH)2D]. 1,25(OH)2D interacts
with its nuclear receptor (VDR) in the intestine and bone in order to maintain
calcium homeostasis. The VDR is also present in a wide variety of other tissues.
1,25(OH)2D interacts with these receptors to have a multitude of important
physiological effects. In addition, it is now recognized that many tissues,
including colon, breast and prostate, have the enzymatic machinery to produce
1,25(OH)2D. The insights into the new biological functions of 1,25(OH)2D in
regulating cell growth, modulating the immune system and modulating the
renin-angiotensin system provides an explanation for why diminished sun exposure
at higher latitudes is associated with increased risk of dying of many common
cancers, developing type 1 diabetes and multiple sclerosis, and having a higher
incidence of hypertension. Another calciotropic hormone that is also produced in
the skin, parathyroid hormone-related peptide, is also a potent inhibitor of
squamous cell proliferation. The use of agonists and antagonists for PTHrP has
important clinical applications for the prevention and treatment of skin
diseases and disorders of hair growth.
PMID: 12899511 [PubMed - in process]
Immunol Cell Biol. 2002 Aug;80(4):340-5.
1 alpha,25-Dihydroxyvitamin D3 inhibits pro-inflammatory cytokine and chemokine expression in human corneal epithelial cells colonized with Pseudomonas aeruginosa.
Xue ML, Zhu H, Thakur A, Willcox M.
Cooperative Research Centre For Eye Research and Technology, School of
Optometry, University of New South Wales, Sydney, New South Wales, Australia.
The cytokines IL-1 beta, IL-6 and the chemokine IL-8 are key mediators of host
inflammation. 1 alpha,25-Dihydroxyvitamin D3 (VD3) has been shown to regulate
host immune responses in vivo and in vitro. The purpose of this study was to
investigate whether the addition of VD3 to human corneal epithelial cells
colonized with Pseudomonas aeruginosa altered the expression of IL-1 beta, IL-6
and IL-8. An immortalized human corneal epithelial (HCE) cell line was used in
this study. After growth to confluency, HCE cells were challenged with P.
aeruginosa strain 6294 in the presence or absence of 10-6 mol/L VD3 for 4 h, 8 h
and 12 h. Gene expression of IL-1 beta, IL-6 and IL-8 was detected by reverse
transcription-PCR (RT-PCR) from total RNA extracted from HCE cells. Protein
concentrations of IL-1 beta, IL-6 and IL-8 in culture supernatants were measured
by ELISA. Addition of VD3 to HCE cells colonized with P. aeruginosa
significantly inhibited the expression of IL-1 beta and IL-8 mRNA and protein (P
< 0.05). Although the expression of IL-6 mRNA was stimulated at 12 h
post-challenge (P < 0.05), the expression of IL-6 protein was inhibited at all
time points after the addition of VD3. In conclusion, this study demonstrated
that VD3 inhibited the P. aeruginosa-induced expression of IL-1 beta, IL-6 and
IL-8 in HCE cells, suggesting that this vitamin may have the potential to become
a novel anti-inflammatory agent in ocular disease.
PMID: 12121222 [PubMed - indexed for MEDLINE]
Int Arch Allergy Immunol. 2002 May;128(1):33-41.
Regulation of cytokine production in human peripheral blood mononuclear cells and allergen-specific th cell clones by 1alpha,25-dihydroxyvitamin D3.
Rausch-Fan X, Leutmezer F, Willheim M, Spittler A, Bohle B, Ebner C,
Jensen-Jarolim E, Boltz-Nitulescu G.
Institute of Pathophysiology, University of Vienna, Vienna, Austria.
BACKGROUND: The steroid hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol), in
addition to its crucial role in calcium homeostasis, exerts several effects on
the immune system by regulating cell proliferation, differentiation, and
maturation. These effects may be exerted through the control of protooncogenes
and the regulation of cytokine production. METHODS: The influence of calcitriol
on cytokines secretion by human peripheral blood mononuclear cells (PBMC)
isolated from healthy donors, and by allergen-specific T helper (Th) cell clones
was studied. PBMC were cultured for 48 h with phorbol myristate acetate (PMA)
and ionomycin in the presence or absence of calcitriol. Human Th cell clones
were stimulated with either Bet v 1 allergen or anti-CD3 antibodies and PMA.
Cytokines were measured in the supernatants by ELISA, and at single-cell level
by FACS. RESULTS: Calcitriol significantly inhibited the production of IL-2,
IFN-gamma and IL-12 by PBMC, as well as the percentage of CD4+ T cells
containing intracytoplasmic IL-2 and IFN-gamma. Interestingly,
calcitriol-treated PBMC induced the production of IL-10 and IL-5, but not of
IL-4. The effect of calcitriol was maximal at 10(-7) to 10(-9) and noneffective
at 10(-11) M. Calcitriol diminished the secretion of IL-1, TNF-alpha, and MG-CSF
in PBMC. Furthermore, calcitriol also decreased the secretion of IL-2 and
IFN-gamma by Th1 clones, and of IL-4 by Th2 clones. CONCLUSIONS: Our data
strongly support the notion that calcitriol modulates the production of
cytokines in a time- and concentration-dependent manner, and suggest that
nonhypercalcemic derivatives of 1alpha,25-dihydroxyvitamin D(3) may be used for
new immunosuppressive therapies. Copyright 2002 S. Karger AG, Basel
PMID: 12037399 [PubMed - indexed for MEDLINE]
J Cell Biochem. 2003;88(2):223-6.
Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators.
Van Etten E, Decallonne B, Verlinden L, Verstuyf A, Bouillon R, Mathieu C.
Laboratory for Experimental Medicine and Endocrinology, Catholic University of
Leuven, Leuven, Belgium.
The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its
classical effects on calcium and bone, for its pronounced immunomodulatory
effects that are exerted both on the antigen-presenting cell level as well as
directly on the T lymphocyte level. In animal models, these immune effects of
1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even
recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3)
in clinical immune therapy are the adverse side effects on calcium and on bone.
TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural
1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with
enhanced in vitro and in vivo immunomodulating capacity compared to the
mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in
redirecting differentiation and maturation of dendritic cells and in inhibiting
phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced
potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes
in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets
grafts, both alone and in combination with cyclosporine A, in overtly diabetic
NOD mice. Moreover, these in vivo effects of TX527 are obtained without the
adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore
that TX527 is a potentially interesting candidate to be considered for clinical
intervention trails in autoimmune diseases. J. Cell. Biochem. 88: 223-226, 2003.
Copyright 2002 Wiley-Liss, Inc.
PMID: 12520518 [PubMed - in process]
FASEB J. 2001 Dec;15(14):2579-85.
Vitamin D: its role and uses in immunology.
Deluca HF, Cantorna MT.
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. email@example.com
In recent years there has been an effort to understand possible noncalcemic roles of vitamin D, including its role in the immune system and, in particular, on T cell-medicated immunity. Vitamin D receptor is found in significant concentrations in the T lymphocyte and macrophage populations. However, its highest concentration is in the immature immune cells of the thymus and the mature CD-8 T lymphocytes. The significant role of vitamin D compounds as selective immunosuppressants is illustrated by their ability to either prevent or markedly suppress animal models of autoimmune disease. Results show that 1,25-dihydroxyvitamin D3 can either prevent or markedly suppress experimental autoimmune encephalomyelitis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease. In almost every case, the action of the vitamin D hormone requires that the animals be maintained on a normal or high calcium diet. Possible mechanisms of suppression of these autoimmune disorders by the vitamin D hormone have been presented. The vitamin D hormone stimulates transforming growth factor TGFbeta-1 and interleukin 4 (IL-4) production, which in turn may suppress inflammatory T cell activity. In support of this, the vitamin D hormone is unable to suppress a murine model of the human disease multiple sclerosis in IL-4-deficient mice. The results suggest an important role for vitamin D in autoimmune disorders and provide a fertile and interesting area of research that may yield important new therapies.
PMID: 11726533 [PubMed - indexed for MEDLINE]
Clin Lab Med. 2000 Sep;20(3):569-90.
Calcium and vitamin D. Diagnostics and therapeutics.
Department of Medicine, Boston University School of Medicine, Massachusetts, USA.
Vitamin D is neither a vitamin nor a nutrient if adequate exposure to sunlight is available to produce adequate quantities of vitamin D3 in the skin. It is well known that an adequate supply of vitamin D, either from the diet or from the skin, is important for maximum bone health throughout life. The new revelation that 25(OH)D can be metabolized to 1,25(OH)2D in the colon, prostate, and skin opens a new chapter in the vitamin D story. It is quite possible that there are two levels of vitamin D sufficiency. One level requires that the serum 25(OH)D levels be at least 20 ng/mL to satisfy the body's requirement for the renal production of 1,25(OH)2D that regulates calcium absorption, and bone calcium mobilization and bone mineralization. The second level may need higher circulating levels of 25(OH)D for maximum cellular health because of the conversion of 25(OH)D to 1,25(OH)2D in extrarenal tissues, such as the prostate, colon, and skin.
PMID: 10986622 [PubMed - indexed for MEDLINE]
Oncol Rep. 2000 Sep-Oct;7(5):1069-74.
Modifying effect of tuna orbital oil rich in docosahexaenoic acid and vitamin D3 on azoxymethane-induced colonic aberrant crypt foci in rats.
Kohno H, Yamaguchi N, Ohdoi C, Nakajima S, Odashima S, Tanaka T.
Department of Serology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. firstname.lastname@example.org
The modifying effect of dietary tuna (Thunnus thynnus orientalis) orbital oil rich in docosahexaenoic acid (DHA) and vitamin D3 (VD3) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACF. The rats were fed the experimental diet containing 5% tuna orbital oil (low fish oil), 23.5% tuna orbital oil (high fish oil), 5% corn oil (low corn oil) or 23.5% corn oil (high corn oil) for 5 weeks, starting 1 week before the first dose of AOM. Animals were sacrificed 2 weeks after the last AOM injection to count colonic ACF and assay the expression of cyclooxygenase (COX)-1 and -2. High corn oil diet significantly increased the development of ACF, when compared with low corn oil diet (P<0.005). High fish oil diet also increased ACF formation compared with low fish oil diet (P<0.01), but the increase was smaller than high corn oil diet. The frequency of ACF was significantly lower in the rats fed high fish oil diet than high corn oil diet (P<0.02). Moreover, frequency of ACF consisted of 4 or more crypts in rats fed the high fish oil diet was significantly lower than that of rats given high corn oil diet. COX-1 and COX-2 expression did not significantly differ among the groups. These results suggest that fish oil derived from tuna, which contains high amounts of DHA and VD3, suppresses the formation and growth of ACF without affecting COX-1 and COX-2 expression, and may have a preventive effect on colon carcinogenesis.
PMID: 10948340 [PubMed - indexed for MEDLINE]
Ann N Y Acad Sci. 1999;889:107-19.
Calcium and vitamin D. Their potential roles in colon and breast cancer prevention.
Garland CF, Garland FC, Gorham ED.
Department of Family and Preventive Medicine, University of California, San Diego 92093, USA. email@example.com
The geographic distribution of colon cancer is similar to the historical geographic distribution of rickets. The highest death rates from colon cancer occur in areas that had high prevalence rates of rickets--regions with winter ultraviolet radiation deficiency, generally due to a combination of high or moderately high latitude, high-sulfur content air pollution (acid haze), higher than average stratospheric ozone thickness, and persistently thick winter cloud cover. The geographic distribution of colon cancer mortality rates reveals significantly low death rates at low latitudes in the United States and significantly high rates in the industrialized Northeast. The Northeast has a combination of latitude, climate, and air pollution that prevents any synthesis of vitamin D during a five-month vitamin D winter. Breast cancer death rates in white women also rise with distance from the equator and are highest in areas with long vitamin D winters. Colon cancer incidence rates also have been shown to be inversely proportional to intake of calcium. These findings, which are consistent with laboratory results, indicate that most cases of colon cancer may be prevented with regular intake of calcium in the range of 1,800 mg per day, in a dietary context that includes 800 IU per day (20 micrograms) of vitamin D3. (In women, an intake of approximately 1,000 mg of calcium per 1,000 kcal of energy with 800 IU of vitamin D would be sufficient.) In observational studies, the source of approximately 90% of the calcium intake was vitamin D-fortified milk. Vitamin D may also be obtained from fatty fish. In addition to reduction of incidence and mortality rates from colon cancer, epidemiological data suggest that intake of 800 IU/day of vitamin D may be associated with enhanced survival rates among breast cancer cases.
PMID: 10668487 [PubMed - indexed for MEDLINE]
Gen Comp Endocrinol. 1995 Jul;99(1):35-40.
Vitamin D3 intoxication in naked mole-rats (Heterocephalus glaber) leads to hypercalcaemia and increased calcium deposition in teeth with evidence of abnormal skin calcification.
Buffenstein R, Laundy MT, Pitcher T, Pettifor JM.
Department of Physiology, Medical School of the Univesity of the Witwatersrand, Johannesburg, South Africa.
Naked mole-rats have no access to obvious sources of vitamin D and therefore have an impoverished vitamin D status. In an investigation into the effects of vitamin D supplementation, inadvertently supraphysiological doses of 130,000 times the normal dose of vitamin D were administered. Within 5 days animals appeared lethargic, with reduced food intake. All but one of the seven animals were killed and blood was collected. Plasma vitamin D metabolites 25(OH)D and 1,25(OH)2D and calcium were determined. Both vitamin D metabolite concentrations exceeded the upper limits of sensitivity of the assays (> 100 ng/ml 25(OH)D and > 210 pg/ml 1,25(OH)2D). Active calcium uptake in the intestine was evident along with concomitant increases in calcium concentration in plasma, bone, and teeth. The remaining animal survived, but showed scab-like formations in the skin around the lower jaw and along the nipple line. X-ray analyses revealed calcium deposition in these cornified regions, although there was no evidence of metastatic calcification in other tissues. Deposition of excess calcium in skin that is regularly sloughed off and in teeth that are continuously worn down and replaced may reduce the vitamin D-induced hypercalcaemia and thus alleviate the effects of vitamin D intoxication.
PMID: 7657155 [PubMed - indexed for MEDLINE]
1: Int J Vitam Nutr Res Suppl. 1989;30:81-6.
High-dose vitamin D therapy: indications, benefits and hazards.
There are two sources of vitamin D available to man: The more important source is the cholecalciferol (vitamin D3), which is produced photochemically in the skin from the provitamin, 7-dehydrocholesterol; vitamin D ingested with food is of secondary importance, but assumes a critical role when an individual is deprived of solar exposure. Vitamin D therefore is not strictly a vitamin. A deficiency of vitamin D ultimately results in osteomalacia in adults and rickets in children, and provision of sunlight or small oral doses of the vitamin can cure this bone condition. There are, however, many less common conditions in which small doses of the vitamin are ineffective, whereas larger doses of vitamin D can achieve healing of the bone disease. These conditions are collectively called vitamin D-resistant diseases and include hypoparathyroidism, genetic and acquired hypophosphataemic osteomalacias, renal osteodystrophy, vitamin D-dependent rickets, and the osteomalacia associated with liver disease and intestinal malabsorption. Unfortunately, large doses of vitamin D continue to be prescribed for a wide variety of diseases in which there is little scientific evidence of their efficacy. The benefits and dangers of high doses of vitamin D are discussed and the problems arising from inappropriate or poorly supervised treatment with vitamin D presented. The serum concentration of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D is increased in certain disease states, and the pathophysiology of some these diseases are presented. The exciting developments in tumour differentiation and the role of high doses of 1,25 dihydroxyvitamin D for the control of leukaemia and other blood and skin diseases are discussed.
PMID: 2507709 [PubMed - indexed for MEDLINE]
Veterinary Clin North Am Exot Anim Pract. 1999
Nutrition of captive reptiles.
Donoghue S, McKeown S.
Nutrition Support Services, Inc., Walkabout Farm, Pembroke, Virginia, USA.
In reptile practice, most nutritional problems arise from improper husbandry,
including poor feeding management and provision of imbalanced diets. Each
reptile species has its own requirements for temperature and humidity, space and
social interaction, lighting, and habitat components. Failure, to provide these
requirements often results in failure to thrive and secondary nutritional
disorders. Common dietary problems include deficiencies of energy, calcium,
vitamin D3, vitamin A, and fiber.
PMID: 11228696 [PubMed - indexed for MEDLINE]
Acta Physiol Hung. 1995;83(1):101-3.
Effects of vitamin D3 administration on the levels of serum calcium and
inorganic phosphorus in the smooth water snake, Enhydris enhydris (Schneider).
Kagwade MV, Pangaonkar AS.
Department of Zoology, B.N.N. College, Bhiwandi, India.
Effect of vitamin D3 administration (12,000 IU/100 g body wt.) on levels of
serum calcium and inorganic phosphorus levels in the smooth water snake,
Enhydris enhydris was investigated. Hypercalcemia and hyperphosphatemia was
observed from day one till the end of the experiment (day 14). Maximum values
were recorded on the 4th day followed by a steady decline.
PMID: 7660831 [PubMed - indexed for MEDLINE]
J Nutr Sci Vitaminol (Tokyo). 1992;Spec No:79-83.
Evolutionary biology and pathology of vitamin D.
Vitamin D, Skin and Bone Research Laboratory, Boston University School of
There is mounting evidence that essentially all fungi, plants and animals living
on earth produce provitamin D. It is likely that once exposed to sunlight, these
provitamins are converted to previtamin D. It is unclear why fungi,
phytoplankton, zooplankton and plants have the capacity to produce such large
quantities of provitamin D. It is likely, however, that provitamin D and
possibly vitamin D play an important biologic role in these organisms. Buchala
and Schmid found, for example, that vitamin D3 promoted adventitious root
development. It may be that provitamin D has a more fundamental function in
lower life forms. Provitamin D and its photoproducts have UV absorption spectra
that overlap with the ultraviolet absorption spectra from ultraviolet
radiation-sensitive macromolecules including DNA, RNA and proteins. Thus,
provitamin D and photoisomers could serve as a photon sink, and therefore, act
as a natural sunscreen to protect lower life forms from the damaging effects of
the high energy ultraviolet radiation that they are exposed to. It is more
clear, however, that amphibians, reptiles, birds, mammals and humans all require
vitamin D and that the vitamin D must be metabolized to 1,25(OH)2D3 before it
can carry-out its physiologic functions on calcium and bone metabolism. The
intense research activities during the past decade on the antiproliferative and
differentiation activities of 1,25(OH)2D3 has opened a new chapter for this
vitamin/hormone. 1,25(OH)2D3 and its analogs are being developed for the
treatment of psoriasis, breast cancer, and leukemia.(ABSTRACT TRUNCATED AT 250
PMID: 1297827 [PubMed - indexed for MEDLINE]
Acta Physiol Hung. 1987;70(4):375-7.
Calcaemic responses in the yellow monitor, varanus flavescens to vitamin D3
Swarup K, Pandey AK, Srivastav AK.
Department of Zoology, University of Gorakhpur, India.
The effect of a daily intramuscular injection of vitamin D3 (2000 IU/100 g b.wt)
on serum calcium level was investigated in Varanus flavescens. This treatment
evoked hypercalcaemia on day 3 which progressed up to day 7. At day 14 a decline
was noticed in the serum calcium level which was followed by a rise from day 21
to day 28.
PMID: 2830765 [PubMed - indexed for MEDLINE]
J Physiol (Paris). 1986;81(1):17-8.
Serum calcium level of freshwater snake, Natrix piscator, in response to vitamin D3 administration.
Srivastav AK, Srivastav SP, Srivastav SK, Swarup K.
The effect of i.m. injection of vitamin D3 (25 IU/100 g b.wt) on serum calcium
level was investigated in Natrix piscator. This treatment evokes hypercalcemia
at day 3 which progresses up to day 5. Thereafter, a decline was observed in the
serum calcium level at day 10 and day 15.
PMID: 3020236 [PubMed - indexed for MEDLINE]