QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Green, M. H. Articles by Green, J. B. Search for Related Content PubMed PubMed Citation Articles by Green, M. H. Articles by Green, J. B.

---

Articles

Increased Rat Mammary Tissue Vitamin A Associated with Increased Vitamin A Intake during Lactation Is Maintained after Lactation^{1 ,2}

Nutrition Department, The Pennsylvania State University, University Park, Pennsylvania 16802

³To whom correspondence should be addressed at Penn State University, Nutrition Department, S-126 Henderson Building South, University Park, PA 16802. E-mail: mhg{at}psu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Although increases in dietary vitamin A increase milk vitamin A, little is known about effects of vitamin A intake on mammary tissue vitamin A levels during and after the reproductive cycle. First, we measured vitamin A concentrations in milk, mammary tissue and liver of lactating rats fed 0, 4, or 50 µmol of vitamin A/kg diet during pregnancy and through d 12 of lactation. Liver vitamin A concentration was significantly affected by diet in lactating females and pups 12 d after parturition. Milk vitamin A concentrations were significantly higher (7.1 ± 2.2 µmol/L, n = 8) in dams fed 50 µmol/kg than in those fed 0 or 4 µmol/kg (1.9 ± 0.3, n = 5 and 2.9 ± 0.7 µmol/L, n = 7; P < 0.001), as were mammary tissue vitamin A concentrations (5.1 ± 1.1 versus 2.2 ± 0.4 and 2.4 ± 0.6 nmol/g; P < 0.001). Next, we maintained female rats on 50 or 10 µmol vitamin A/kg diet during pregnancy and lactation and then on 4 µmol/kg diet after pups were weaned on d 21. On d 21, mammary tissue vitamin A concentrations were 3.14 ± 0.75 versus 1.52 ± 0.21 nmol/g in dams fed 50 versus 10 µmol/kg (n = 4/group; P < 0.001). Mammary tissue vitamin A concentrations were not significantly affected by time from 7 to 49 d after lactation and averaged 8.5 ± 0.4 and 4.9 ± 0.8 nmol/g on d 49 in dams fed 50 versus 10 µmol/kg (n = 4; P < 0.001). We conclude that diet-induced differences in rat mammary tissue vitamin A developed during pregnancy and lactation are maintained for 7 wk after lactation.

KEY WORDS: • vitamin A intake • milk • lactation • mammary tissue • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Severalstudies indicate that the supplementation of lactating women with vitamin A results in increased concentrations of vitamin A in milk. Villard and Bates (1) reported that milk vitamin A levels were increased in poorly nourished women who consumed 2.3 µmol vitamin A/d during pregnancy and lactation. More recently, Roy et al. (2) found that breast milk vitamin A levels were increased 283% in women who received a supplement of vitamin A (209 µmol) at delivery. Davila et al. (3) found that milk vitamin A concentration was doubled in lactating rats fed 52 versus 2.1 µmol of vitamin A/kg diet during pregnancy and lactation.

Although it seems clear that increases in vitamin A intake increase the vitamin A concentration of milk, little is known about effects of dietary vitamin A on mammary tissue vitamin A content either in humans or in animal models. In a recent case-control study, Zhang et al. (4) found a positive but nonsignificant correlation between vitamin A levels in breast adipose tissue and vitamin A intake in women. Here, we investigated the effects of vitamin A intake during pregnancy and lactation on mammary tissue vitamin A levels in rats, hypothesizing that higher vitamin A intakes would lead to higher vitamin A concentrations in mammary tissue as well as in milk. In addition, we measured mammary tissue vitamin A levels as a function of time after lactation in rats fed two levels of dietary vitamin A during pregnancy and lactation, hypothesizing that diet-induced increases in mammary tissue vitamin A would be maintained after the end of lactation. Potential effects of dietary vitamin A on mammary tissue vitamin A levels are of interest in view of the finding that women with higher vitamin A intakes have a lower risk of breast cancer (5 6 7) .

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Animals and diets.

Female (60-d-old) and adult male Sprague-Dawley rats were purchased from Harlan Teklad (Madison, WI). Rats were individually housed at 22–24°C, 50% humidity, with light from 0700 to 1900 h. Animals had free access to food (see later) and water throughout the study; females were weighed twice weekly. Animal procedures were approved by The Pennsylvania State University’s Animal Care and Use Committee.

Female rats were fed a modification of the AIN-93G diet (8) containing 200 g vitamin-free casein, 397 g cornstarch, 132 g maltodextrin, 100 g sucrose, 50 g cellulose, 35 g mineral mix (AIN-93G-MX; Teklad), 10 g vitamin A–free vitamin mix (TD94161; Teklad), 3 g L-cystine, 2.4 g choline bitartrate, 0.014 g t-butylhydroquinone and 70 g soybean oil per kg diet, to which had been added 10 µmol of retinyl palmitate (Sigma Chemical Co., St. Louis, MO) per kg of diet. Male rats were fed the same diet when they were being used for breeding and were fed a commercial cereal-based diet (Laboratory Rodent Diet 5001; PMI Nutrition International, St. Louis, MO) at other times.

Experiment 1.

Female rats were mated beginning at age 63 d by housing two females with one male for 5 d. After mating, females were randomly assigned to one of three dietary groups that consumed the purified diet containing 0, 4 or 50 µmol retinyl palmitate/kg. The 4 µmol vitamin A/kg diet was chosen to provide a vitamin A intake of 60 nmol/d (a slight positive balance) or 170 nmol /(kg ^0.75 · d), assuming a body weight of 250 g and a food intake of 15 g/d. In comparison, the amount recommended for lactating women in the United States is 161 nmol /(kg ^0.75 · d) (9) . The 50 µmol/kg diet provided 2121 nmol /(kg ^0.75 · d), or 13 times the recommended dietary intake for women, an amount that may be obtained from dietary supplements.

Two to 3 d after parturition, litter sizes were reduced to eight pups per dam. On d 12 of lactation, the pups were removed, and the dams were anesthetized with ketamine HCl/xylazine [100 mg ketamine/kg body weight (Aveco, Fort Dodge, IA) and 10 mg xylazine/kg (Mobay, Shawnee, KS)]. Oxytocin (21 IU; Sigma Chemical Co.) was injected intramuscularly, and milk (500 µL) was obtained using gentle suction during manual massage of the mammary glands. Samples were aliquoted for subsequent analysis of vitamin A and lactose and frozen at -60°C under nitrogen. Mammary tissue was dissected using a No. 10 scalpel, weighed, flash-frozen in liquid nitrogen and stored under nitrogen at -60°C for later analysis of vitamin A and lactose. Then, the whole body was perfused with Hanks’ balanced salt solution, pH 7.2; livers were excised, weighed, frozen, lyophilized and then stored under nitrogen at -16°C for subsequent vitamin A analysis. Pups were weighed and then killed by asphyxiation with carbon dioxide; livers were removed, weighed, frozen, lyophilized and then stored under nitrogen at -16°C for vitamin A analysis. At the time of analysis, pup livers from each litter were pooled.

Experiment 2.

Female rats were mated as described above between 69 and 83 d of age. After mating, females remained on the diet providing 10 µmol vitamin A/kg (150 nmol/d) or were fed a higher amount of vitamin A (50 µmol/kg or 750 nmol/d).

At 3 d after parturition, litter sizes were adjusted to seven pups per dam. At 21 d after parturition, pups were weighed and killed by asphyxiation with carbon dioxide. A milk sample was obtained from four dams per dietary group, and then these females were killed as described for dams earlier; mammary tissue was dissected and livers were excised. Tissues were weighed and stored under nitrogen at -16°C for later analysis of vitamin A. Remaining females were moved to stainless steel hanging cages on d 21 and were fed the purified diet containing 4 µmol vitamin A/kg. Females (four per group) were killed at d 7, 14, 28 and 49 after the end of lactation, and livers and mammary tissue were removed and frozen for vitamin A analysis.

To assess baseline levels of vitamin A in liver and mammary tissue, four rats from each group that did not conceive were killed at 103 d of age, when rats mated at the same time were giving birth. Livers and samples of mammary tissue were obtained as described earlier.

Vitamin A analyses.

Analyses were conducted under fluorescent lights shaded with a UV-blocking film (CLCH; Sydlin, Lancaster, PA). An internal standard of the nonsaponifiable retinoid, all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1-ol [TMMP-retinol⁵ ); donated by Hoffmann-La Roche, Basel, Switzerland] was added to aliquots of lyophilized liver (3 x 0.15 g), mammary tissue (5 x 0.5 g) and milk (100 µL). Samples were saponified and extracted using procedures described by Thompson et al. (10) and Green and Green (11) . Retinol and TMMP-retinol in lipid extracts were separated by reverse phase HPLC (series 1050; Hewlett Packard, Wilmington, DE) using a Supelcosil 3-µm LC-18 column and guard column (Supelco, Bellefonte, PA) with UV detection at 325 nm and a mobile phase of methanol/water [91:9 (v/v)] at 1.5 mL/min. Peak areas were calculated using a Hewlett-Packard 1050 Chemstation, and retinol mass was determined by an internal standard method, using the mass-to-area ratios for TMMP-retinol and retinol standard curves.

Within-animal variability in analyses of vitamin A in mammary tissue was much higher than those for liver. Specifically, the mean coefficient of variation for vitamin A concentration in the three replicates of liver analyzed for each dam (n = 44) in expt. 2 was 4.0% (range 0.36–14.3%). In contrast, variation among the five replicates of mammary tissue was much higher (mean 17.5%, range 4.2–39.2%), although both tissues were analyzed by the same investigator using the same procedures (except that livers were freeze-dried). For cases in which the coefficient of variation for mammary tissue vitamin A was >20%, analyses were repeated. We speculate that the higher variation in analyses of vitamin A in mammary tissue was due to the difficulty in obtaining a representative sample of a heterogeneous tissue and possibly the distribution of various cell types and their contribution to tissue vitamin A levels.

Lactose analysis.

To determine whether residual milk in mammary tissue contributed substantially to the measured amount of vitamin A in mammary tissue in expt. 1, lactose, a sugar unique to milk, was determined in milk and mammary tissue homogenates with an enzymatic assay (12) . Based on milk lactose concentration (93 ± 35 nmol/L, n = 19), we estimated the amount of residual milk in the mammary glands from the measured amount of lactose in mammary tissue. Because vitamin A in residual milk contributed only 2.2–3.9% of the observed mammary tissue vitamin A content, we did not correct observed mammary tissue vitamin A data for residual milk.

Statistical analyses.

Data are presented as means ± SD. Statistical analyses were conducted with analysis of variance and Tukey’s post hoc test (13) or independent t tests with an level of 0.05. When variance was unequal among groups, data were log-transformed before analysis of variance was conducted. Pearson’s correlations (13) were calculated to determine whether correlations existed between vitamin A intake and the dependent variables.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Experiment 1.

There were no significant effects of dietary vitamin A on maternal body weights (data not shown), liver weight or mammary tissue weight on d 12 of lactation (Table 1 ). However, vitamin A intake had a significant effect on liver vitamin A concentration. Concentrations were three times higher in dams fed the higher vitamin A load than in those fed 0 µmol vitamin A/kg and 1.7 times higher than in those fed 4 µmol vitamin A/kg (Table 1) . Maternal dietary vitamin A level also had a significant effect (P < 0.001) on vitamin A concentrations in pup liver: concentrations were 160 ± 15.9 nmol/g (n = 5 litters) in offspring of females fed 50 µmol vitamin A/kg versus 46.5 ± 11.3 nmol/g in 2 litters from dams fed 0 and 59.1 ± 4.6 nmol/g in 5 litters from dams fed 4 µmol/kg. In a related experiment (14) , plasma retinol concentrations were the same in lactating dams fed 50 and 10 µmol vitamin A/kg diet. Based on those results and conventional wisdom, we assume that plasma retinol concentrations were normal in all groups in the current study.

Overall, results of expt. 1 indicate that as is the case for milk, mammary tissue vitamin A concentrations are increased in lactating rats in response to physiologically reasonable increases in vitamin A intake. Our findings complement the results of a short-term study by Ross et al. (15) . In those experiments, 15–30% of radioactivity injected as [³H]vitamin A–labeled chylomicrons was recovered in mammary glands of lactating rats 2–3 min after the administration (A. C. Ross, Penn State University; personal communication), suggesting that newly absorbed vitamin A is taken up by lactating mammary tissue before hepatic processing. Based on those results, our current data and information from related work (14) , we hypothesize that the increase in mammary tissue vitamin A in lactating rats fed the high vitamin A–containing diet is due to a targeting of chylomicron vitamin A (retinyl esters) to mammary tissue. Assuming that all of the vitamin A in mammary tissue and milk of rats fed the vitamin A–free diet in expt. 1 is derived from retinol carried by retinol-binding protein, we estimate that at the minimum, chylomicrons may be supplying 60% of the mammary tissue vitamin A in lactating rats fed 50 µmol vitamin A/kg in the current experiment.

A potentially interesting observation was made on data from expt. 1. Although the ratio of vitamin A concentration in milk to liver (x100) was almost the same in all groups (0.560 ± 0.170, 0.525 ± 0.134 and 0.540 ± 0.249 in 0, 4 and 50 µmol/kg groups), the ratio of mammary tissue to liver vitamin A concentrations was significantly affected by diet (P < 0.05), averaging 0.627 ± 0.135, 0.538 ± 0.202 and 0.390 ± 0.126, respectively. If indeed the ratio of vitamin A concentration in milk versus liver is constant over a wide range of chronic vitamin A intakes, one should be able to predict liver vitamin A concentration based on analysis of milk retinol. Such an assessment tool would be simple to perform in the field, although its application would be limited to lactating women. However, as pointed out by Stoltzfus et al. (16) , monitoring the response of lactating women to a vitamin A intervention program may be reflective of the community-wide response. The fact that the ratio of mammary tissue vitamin A to liver vitamin A was affected by vitamin A intake in the current study indicates that at these intake levels and during the time frame of this study, mammary tissue vitamin A concentration is less influenced by dietary vitamin A than is liver concentration.

Experiment 2.

In female rats that did not become pregnant, vitamin A concentrations in liver were significantly higher in rats fed 50 µmol vitamin A/kg diet than in those fed 10 µmol/kg (1156 ± 136 versus 568 ± 53 nmol/g, n = 4/group). In contrast, vitamin A concentrations in mammary tissue of the same rats were not significantly affected by dietary vitamin A intake (8.62 ± 1.03 versus 6.40 ± 0.91 nmol/g; P = 0.090).

Body weights on d 21 of lactation were 307 ± 23 (n = 21) and 300 ± 21 g (n = 19) in dams fed 10 versus 50 µmol vitamin A/kg diet. At that time, pups were weaned and dams were fed 4 µmol vitamin A/kg (chosen to maintain vitamin A balance). Liver vitamin A concentrations were not significantly affected by time from 0 to 49 d after lactation. As expected, there was a significant effect of diet on liver vitamin A concentration, averaging 768 ± 38 versus 2629 ± 40 nmol/g in the 10 versus 50 µmol/kg diet group.

Mammary tissue weights were significantly higher at the time of weaning (20 g) than after lactation (7–9 g; P < 0.001) (Table 2 ) due to involution of the mammary gland after the cessation of lactation. There was no effect of time on mammary tissue weights from d 7 to 49. Diet, time and diet x time interaction terms were all significant for vitamin A concentrations in mammary tissue between d 0 and 49 after lactation (P < 0.001), but the diet x time interaction term was not significant between postlactation d 7 and 49 (P = 0.057). Vitamin A concentration in mammary tissue increased from 0 to 14 d after lactation as involution occurred and then slowly decreased on d 28 and 49. Within each dietary group, mammary tissue vitamin A concentrations were significantly lower at time zero (weaning) than at later times. At all times, mammary tissue vitamin A concentration was significantly higher (70–120%) in dams fed 50 versus 10 µmol vitamin A/kg during pregnancy and lactation. Time and the diet x time interaction were not significant for total mammary tissue vitamin A levels: levels were 34.6 ± 8.8 nmol at weaning versus 39.7 ± 11 nmol at 49 d after lactation in the 10 µmol/kg group and 57.5 ± 33.0 versus 66.0 ± 20.0 nmol in the 50 µmol/kg group. Our results indicate that the diet-induced accumulation of vitamin A in mammary tissue that accompanied pregnancy and lactation was retained for 7 wk after lactation ended, even though vitamin A intake was lower. In comparing rats in the 50 µmol with those in the 10 µmol vitamin A/kg diet group, the 3-fold greater vitamin A intake was associated with an increased mammary tissue vitamin A concentration of 100%, and this difference was maintained for 7 wk after lactation.

	ACKNOWLEDGMENTS

 
We thank A. Catharine Ross (Nutrition Department, Penn State University) for contributions to the development of this research, Ronald S. Kensinger (Department of Dairy and Animal Science, Penn State University) for help in setting up the lactose assay and Sean K. Kelley (current address: Genentech, South San Francisco, CA) for technical contributions to the project.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 96, Washington [Snyder, R. W., Kelley, S. K., Green, M. H. & Ross, A. C. (1996) Dietary vitamin A affects vitamin A content of mammary glands in rats. FASEB J. 10:A525 (abs.)].

² Supported by National Institutes of Health grant RO1HD32500 (to M.H.G.) and a Penn State University interdisciplinary seed grant (to M.H.G. and R. S. Kensinger, Department of Dairy and Animal Science, Penn State University).

⁴ Current address: Chemical Industry Institute of Toxicology, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, NC 27709.

⁵ Abbreviation used: TMMP-retinol, all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1-ol.

Manuscript received October 13, 2000. Revision accepted January 29, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1. Villard L., Bates C. J. Effect of vitamin A supplementation on plasma and breast milk vitamin A levels in poorly nourished Gambian women. Hum. Nutr. Clin. Nutr. 1987;41C:47-58[Medline]

2. Roy S. K., Islam A., Molla A., Akramuzzaman S. M., Jahan F., Fuchs G. Impact of a single megadose of vitamin A at delivery on breast milk of mothers and morbidity in their infants. Eur. J. Clin. Nutr. 1997;51:302-307[Medline]

3. Davila M. E., Norris L., Cleary M., Ross A. C. Vitamin A during lactation: relationship of maternal diet to milk vitamin A content and to the vitamin A status of lactating rats and their pups. J. Nutr. 1985;115:1033-1041

4. Zhang S., Tang G., Russell R. M., Mayzel K. A., Stampfer M. J., Willett W. C., Hunter D. J. Measurement of retinoids and carotenoids in breast adipose tissue and a comparison of concentrations in breast cancer cases and control subjects. Am. J. Clin. Nutr. 1997;66:626-632[Abstract/Free Full Text]

5. Potischman N., McCulloch C. E., Byers T., Nemoto T., Stubbe N., Milch R., Parker R., Rasmussen K., Root M., Graham S., Campbell T. C. Breast cancer and dietary and plasma concentrations of carotenoids and vitamin A. Am. J. Clin. Nutr. 1990;52:909-915[Abstract/Free Full Text]

6. Hunter D. J., Manson J. E., Colditz G. A., Stampfer M. J., Rosner B., Hennekens C. H., Speizer F. E., Willett W. C. A prospective study of the intake of vitamin C, E, and A and the risk of the breast cancer. N. Engl. J. Med. 1993;329:234-240[Abstract/Free Full Text]

7. Rohan T. E., Howe G. R., Friedenreich C. M., Jain M., Miller A. B. Dietary fiber, vitamin A, C, and E, and risk of breast cancer: a cohort study. Cancer Causes Control 1993;4:29-37[Medline]

8. Reeves P. G., Nielsen F. H., Fahey G. C., Jr AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Committee on the Reformulation of the AIN-76A Rodent Diet. J. Nutr. 1993;123:1939-1951

9. Olson J. A. Recommended dietary intakes (RDI) of vitamin A in humans. Am. J. Clin. Nutr. 1987;45:704-716[Abstract/Free Full Text]

10. Thompson J. N., Erdody P., Brien R., Murrary T. K. Fluorometric determination of vitamin A in human blood and liver. Biochem. Med. 1971;5:67-89[Medline]

11. Green M. H., Green J. B. Experimental and kinetic methods for studying vitamin A dynamics in vivo. Methods Enzymol 1990;190:304-317[Medline]

12. Coffey R. G., Reithel F. J. An enzymatic determination of lactose. Anal. Biochem. 1969;32:229-232[Medline]

13. Ryan B. F., Joiner B. L., Ryan T. A., Jr Minitab Handbook 2nd ed. 1985 PWS Publishers Boston, MA.

14. Green, M. H., Green, J. B., Akohoue, S. A. & Kelley, S. K. (2001) Vitamin A intake affects the contribution of chylomicrons versus retinol-binding protein to milk vitamin A in lactating rats. J. Nutr. 131 (in press).

15. Ross A. C., Pasatiempo A.M.G., Green M. H. Chylomicron vitamin A uptake in the lactating mammary gland: possible relationship to retinoids in breast milk and breast. FASEB J 1996;10:A467(abs.)

16. Stoltzfus R. J., Habicht J.-P., Rasmussen K. M., Hakimi M. Evaluation of indicators for use in vitamin A intervention trials targeted at women. Int. J. Epidemiol. 1993;22:1111-1117[Abstract/Free Full Text]

This article has been cited by other articles:

				S. H. Gieng, M. H. Green, J. B. Green, and F. J. Rosales Model-based compartmental analysis indicates a reduced mobilization of hepatic vitamin A during inflammation in rats J. Lipid Res., April 1, 2007; 48(4): 904 - 913. [Abstract] [Full Text] [PDF]

				S. H. Gieng and F. J. Rosales Plasma {alpha}1-Acid Glycoprotein Can Be Used to Adjust Inflammation-Induced Hyporetinolemia in Vitamin A-Sufficient, but Not Vitamin A-Deficient or -Supplemented Rats J. Nutr., July 1, 2006; 136(7): 1904 - 1909. [Abstract] [Full Text] [PDF]

				S. A. Akohoue, J. B. Green, and M. H. Green Dietary Vitamin A Has Both Chronic and Acute Effects on Vitamin A Indices in Lactating Rats and Their Offspring J. Nutr., January 1, 2006; 136(1): 128 - 132. [Abstract] [Full Text] [PDF]

				R. Piantedosi, N. Ghyselinck, W. S. Blaner, and S. Vogel Cellular Retinol-binding Protein Type III Is Needed for Retinoid Incorporation into Milk J. Biol. Chem., June 24, 2005; 280(25): 24286 - 24292. [Abstract] [Full Text] [PDF]

				S. H. Gieng, J. Raila, and F. J. Rosales Accumulation of retinol in the liver after prolonged hyporetinolemia in the vitamin A-sufficient rat J. Lipid Res., April 1, 2005; 46(4): 641 - 649. [Abstract] [Full Text] [PDF]

				A. C. Ross, A. M. G. Pasatiempo, and M. H. Green Chylomicron Margination, Lipolysis, and Vitamin A Uptake in the Lactating Rat Mammary Gland: Implications for Milk Retinoid Content Experimental Biology and Medicine, January 1, 2004; 229(1): 46 - 55. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Green, M. H. Articles by Green, J. B. Search for Related Content PubMed PubMed Citation Articles by Green, M. H. Articles by Green, J. B.