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 Raj, T. Articles by Kurpad, A. V. Search for Related Content PubMed PubMed Citation Articles by Raj, T. Articles by Kurpad, A. V.

---

Nutrient Requirements and Optimal Nutrition

Intestinal Microbial Contribution to Metabolic Leucine Input in Adult Men^1,2

³ St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India and ⁴ State University of New York, Stony Brook, NY 11790

^* To whom correspondence should be addressed. E-mail: a.kurpad{at}iphcr.res.in.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
New estimates of the indispensable amino acid requirements of adult humans are much higher than previously thought and questions the adequacy of cereal-based diets of low protein quality. However, dietary amino acid requirements may be supplemented by contributions from the intestinal microbiota. This study measured the contribution of intestinal microbes to leucine input in healthy adult men. Fourteen adult men were studied during each of 2 11-d periods (before and after intestinal antimicrobial treatment), in which leucine was supplied at 1.25 times the estimated average requirement (EAR) (d 1–7) and at 2.5 times the EAR (d 8–11) providing an L-amino acid diet. We estimated fasting- and fed-state leucine oxidation on d 7 and d 11 using a ¹³C-leucine tracer infusion. The microbial contribution to body leucine input was calculated from the relationship of leucine oxidation to leucine intake and the reduction in leucine oxidation after antimicrobial treatment. Antimicrobial treatment did not affect the slope of the relationship of leucine oxidation to leucine intake. Mean and fed-state leucine oxidation declined by 13 and 20%, respectively (both P < 0.05) after antimicrobial treatment with the 1.25 EAR diet, but not with the 2.5 EAR diet. The contribution of the intestinal microbiota to body leucine input was estimated to be between 19 and 22% at the 1.25 EAR diet. The contribution of the intestinal microbiota to body amino acid homeostasis may be significant at maintenance intakes, but its long-term nutritional importance remains to be determined.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

It is conventionally assumed that all the essential amino acid requirements of humans (and other nonruminants) must be supplied by the diet. However, there is evidence of substantial absorption of essential amino acids synthesized by the gut microbiota in simple-stomached animals and humans (6,7). From labeling experiments using ¹⁵NH₄Cl incorporation into microbial lysine in normal human subjects and ileostomates, it has been estimated that microbial lysine absorption was 29–68 mg·kg^–1.d^–1, which is of the same order as the estimated average requirement (EAR)⁵ of lysine (30 mg·kg^–1.d^–1) in human adults (8). However, although this suggests that nutritionally relevant amounts of microbially derived lysine are absorbed, quantifying the amount absorbed is technically complicated by the need to estimate the ¹⁵N enrichment of the lysine being absorbed, which seems to occur mostly in the small intestine (9,10). In normal humans, estimates of microbial lysine absorption based on the ¹⁵N-lysine enrichments of fecal microbes are almost certainly incorrect, because there is continued incorporation of ¹⁵N from ammonia into microbial amino acids in the colon, from which there is little absorption (9,10). Estimates based on the ¹⁵N-lysine enrichments of microbes in ileostomy effluent is also unlikely to be correct, because a consequence of ileostomy is an increase in microbial numbers and an alteration of the constituent species of the ileal microflora (11). These uncertainties make the interpretation of such ¹⁵N-labeling studies difficult. Nevertheless, the fact that there is some, albeit poorly quantified, absorption of microbially synthesized amino acids leads to the concept of a metabolic amino acid supply, which is the sum of the dietary supply and the contribution of the intestinal microbiota.

Because the amounts of amino acids supplied by the intestinal microbial route have not been satisfactorily quantified, we devised a method based on the linear relationship between leucine oxidation and leucine intake at intakes that are well above the daily leucine requirement level of 40 mg·kg^–1.d^–1 (12). If the total metabolic leucine input into the body is considered as the sum of dietary and microbially derived leucine, then by reducing the numbers of intestinal microbes, the leucine input into the body would be expected to decrease. This decline in leucine oxidation would then allow the microbially derived leucine supply to be estimated from the slope of the relationship between leucine intake and oxidation. The calculation of microbially derived leucine input would also allow estimation, based on the amino acid composition of microbial proteins, of the input of other indispensable amino acids by this route. This study was therefore designed to quantify the microbially derived leucine input in healthy South Indian men.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Fourteen otherwise healthy adult male subjects in a normal BMI range participated in this experiment. Subjects were screened using a medical history, physical examination, blood cell count, and a routine biochemical profile that included fasting blood sugar, liver function tests, and serum urea and creatinine. The subjects were weighed to the nearest 0.1 kg and their height was measured to the nearest 1.0 cm. The logarithm of the sum of 4 (biceps, triceps, subscapular, and suprailiac) skinfolds was used in age- and gender-specific equations (13) to obtain an estimate of body density, from which body fat and fat-free mass were estimated (14) (Table 1). The purpose of the study and the potential risks involved were explained to each subject and a written informed consent was obtained from each subject. The Institutional Ethical Review Board of St. John's Medical College approved the research protocol.

The primed i.v. ¹³C-leucine approach was used, with indirect calorimetry, blood, and breath sampling as previously described (15). The total duration of the tracer infusion was 18 h, which began at 0000 and ended at 1800. Briefly, 1-¹³C-leucine (99.0 atom%; Cambridge Isotope Laboratories) was given as a primed, constant i.v. infusion at a known rate of 2.8 µmol·kg^–1.h^–1 (the prime was 4.2 µmol/kg). The bicarbonate pool was primed with 0.8 µmol/kg of ¹³C-sodium bicarbonate (99 atom%; MassTrace). Baseline blood and breath samples were collected at –30, –15, and –5 min before the infusion began. Blood and breath samples were collected every 30 min during the last 120 min of the fasting period (0400–0600) and similarly collected every 30 min during the last 90 min of the fed period (1630–1800). The analyses of breath for ¹³CO₂ enrichment by isotope ratio MS (Europa Scientific) and plasma samples for ¹³C--ketoisocaproic acid (KIC) by GC-MS (Varian) were as previously described (15). Leucine oxidation (µmol·kg^–1.30 min^–1) was calculated as the ¹³CO₂ production rate (µmol·kg^–1.30 min^–1) to plasma ¹³C-KIC enrichment (mole percent excess) ratio, whereas the leucine flux was calculated as the ratio of the tracer infusion rate (µmol·kg^–1.30 min^–1) to the plasma ¹³C-KIC enrichment (mole percent excess).

The anthropometric and metabolic variables are presented as arithmetic means ± SD. The study was powered to look for 1-tailed significant differences in leucine oxidation between the 2 phases of the study (because it was expected that no increase in leucine oxidation would occur after antimicrobial therapy) for either leucine intake, assuming a 20% difference between phases, SD that was 25% of the mean, power of 80%, and a 5% level of significance. Therefore, the primary analysis for differences in leucine oxidation between the 2 phases of the study, the 2 leucine intakes, and between metabolic periods (fasting and fed) were the 1-tailed Student's paired t test, because leucine oxidation was expected to increase only with feeding or with increased dietary intake. The data were also subjected to secondary analysis using a 3-level model in which each subject was measured in 2 phases, with 2 leucine intakes and 2 metabolic phases as within-phase variables. We used a pooled regression of leucine oxidation on leucine intake to test for significant differences in slopes between the phases of the study. The slope of the leucine oxidation-leucine intake relationship in the pretreatment phase was used along with the difference in the oxidation of leucine between the pre- and post-treatment phases to calculate the reduction in leucine input into the body when antimicrobials were given. Results were considered significant if P 0.05. The data were analyzed using SPSS (v13.0).

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
The mean BMI was 21 kg/m² and the mean percent body fat in the subjects was 17% (Table 1). The subjects' blood biochemical parameters were within normal limits at recruitment. Their mean erythrocyte sedimentation rate (ESR) was 5.9 ± 4.0 mm/h^. Body weight of the subjects did not differ between the phases of the study. There were no adverse side effects from the antibiotic treatment and none of the subjects reported any symptoms or change in appetite or bowel movements.

Based on the primary analysis (Table 3), the fasting- and fed-state leucine oxidation rates in either the pre- or post-treatment phases did not change after the 1.25 EAR leucine diet was fed; however, these differences were significant after the 2.5 EAR diet was fed (P < 0.001 for both pre- and post-treatment phases). The fed-state leucine oxidation rate was greater with the 2.5 EAR than with the 1.25 EAR leucine diet (P < 0.001, for both treatment phases). With the 1.25 EAR diet, the fed-state leucine oxidation rate was lower after giving antibiotics (P = 0.02). When the entire data set was analyzed by a 3-level model, there was a significant effect of treatment phase within leucine intakes and metabolic phases (P < 0.001). Post-hoc analysis revealed that the 1-tailed significant difference between treatment phases lay in the fed-state oxidation at the 1.25 EAR leucine intake level, as shown in the primary analysis.

The decline in the mean value of the fasting- and fed-state leucine oxidation rates, with the 1.25 EAR diet, was used to assess the microbial contribution to daily body leucine supply. The slope of the leucine oxidation-leucine intake relation did not differ between the pre- and post-treatment phases (Table 3). The mean slope of the relationship between mean leucine oxidation (µmol·kg^–1.30 min^–1) and leucine intake (mg·kg^–1.d^–1) in the pretreatment phase was 0.25 ± 0.16 (µmol·kg^–1.30 min^–1)·(mg·kg^–1.d^–1)^–1. The former, together with the mean difference between the phases in fed-state leucine oxidation with the 1.25 EAR diet (1.5 µmol·kg^–1.30 min^–1), was used to estimate the decrease in leucine input into the body due to antibiotic treatment. These calculations were not made with the 2.5 EAR diet, because there was an effect of treatment phase within leucine intakes (1.25 or 2.5 EAR) and metabolic phase (P < 0.001) and because, at that intake, antibiotics did not reduce the fed-state leucine oxidation rate. The microbially derived leucine input was estimated to be 9.6 mg·kg^–1.d^–1, which represents 19.2% of the leucine intake provided by the 1.25 EAR diet. When the microbial contribution to leucine input into the body was calculated using the mean of the fasting- and fed-state leucine oxidation data for evaluating a possible whole-day contribution, the corresponding value for the mean slope was 0.13 ± 0.09 (µmol·kg^–1.30 min^–1).(mg·kg^–1.d^–1)^–1. From this, the microbially derived leucine input at the 1.25 EAR diet was estimated to be 11.0 mg·kg^–1.d^–1, representing 22.0% of the leucine intake supplied by the 1.25 EAR diet.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Recommendations for the daily requirement for amino acids conventionally assume that all these requirements would come from the diet and that there is no other source of these amino acids. However, microbes within the gastrointestinal tract are capable of hydrolyzing urea and synthesizing essential amino acids. Although the magnitude of the contribution could not earlier be determined with any certainty (8), the present study suggests that the microbial contribution to the supply of leucine was between 19 and 22% of the intake (1.25 EAR), depending on the method of calculation. This corresponds to 24–27% of the EAR (1). By extension, one would expect the microbial supply of other indispensable amino acids to be a broadly similar proportion of requirements.

In previous studies, based on the administration of ¹⁵N ammonium chloride, the microbial contribution to lysine absorption in normal adults and ileostomates was measured from the ¹⁵N enrichment of lysine in plasma and in the microbial fraction of feces or ileostomy fluid, along with measurements of the plasma rate of appearance of ¹⁵N-lysine (8,16). Other studies of fecal microbial and urinary lysine enrichments in adults given lactose-(¹⁵N)₂-ureide suggested an availability of microbial lysine that matched the daily requirement (17), whereas studies on undernourished infants administered ¹⁵N₂-urea, along with the measurement of urinary lysine enrichment and an assumption of the fecal microbial lysine enrichment, suggested de novo lysine synthesis rates that were 10% or more of the daily requirement in these children in whom the rate of urea hydrolysis was high (18). These estimates of the availability of microbially derived lysine were substantial in relation to the dietary requirement, but depend critically on 2 assumptions. The first is that the ¹⁵N enrichment of lysine in the microbial protein that is isolated from digesta is a true measure of the microbial lysine being absorbed. Because there is likely to be continued incorporation of ¹⁵N from ammonia into microbial amino acids in the colon, this value may be very different from that at the sites of absorption, which seem to be mostly in the small intestine (9,10). Although the ileostomates had a lower plasma ¹⁵N-lysine enrichment than the intact subjects given the same amount of ¹⁵N ammonium chloride, the ¹⁵N enrichment of lysine in the microbial protein of ileostomy fluid was less than one-third of that in the fecal microbial protein of intact subjects (8,16); as a result, the estimated microbial lysine uptake was 68 mg·kg^–1.d^–1 or more than twice the requirement. Using the urea enrichment as a surrogate for fecal microbial enrichment is also likely to underestimate microbial lysine availability, because other sources of N are available for microbial protein synthesis in the gut (19,20). The uncertainties about the actual ¹⁵N enrichment of the lysine being absorbed make the precise interpretation of these ¹⁵N-labeling experiments difficult. In fact, the same problem exists for other tracers, such as ¹⁴C (9).

We therefore devised an alternative approach to measure the contribution of indispensable amino acids from the intestinal microflora, based on the linear relationship between the change in leucine intake and the change in oxidation, at leucine intakes that are above the daily requirement (12). Our reasoning was that if there were an appreciable microbial leucine input into the body, that would be reduced by the suppression of intestinal microbes, leading to a decrease in the metabolic leucine input into the body with a corresponding decrease in leucine oxidation. This decrease in leucine oxidation could then be used to predict the amount of microbially derived leucine from the slope of the previously defined relationship between leucine intake and oxidation. We compared the slopes of this relationship before and after antimicrobial treatment to assess if the treatment had any effect on this relationship. The fact that there was no significant change in pooled slopes suggests that there was no systematic effect of the antibiotics on leucine kinetics.

Previous estimates of the contribution of microbial amino acids to the host's needs (7–10) involve a 2nd assumption: that microbial amino acids are entirely synthesized de novo, using nitrogen that would otherwise be of little or no nutritional value, such as urea or ammonia. Recent studies of pigs (19,20) given simultaneous infusions of L-[1-¹³C]-valine and [¹⁵N¹⁵N]-urea suggest that only a small proportion of valine in microbial protein is synthesized de novo using N from urea; a larger proportion is preformed valine from endogenous secretions (detected as [1-¹³C]-valine) and the largest proportion, by difference, was assumed to be preformed valine from the diet. Thus, although estimates of microbial lysine absorption may be correct, they do not represent a net gain of amino acid but, to the extent that microbial amino acids from dietary or endogenous sources are incorporated intact into microbial protein that is later digested, they simply describe an additional step in the route by which those amino acids reach the host.

By contrast, the present approach attempts to assess the net benefit to the host, avoiding the assumptions required by previous approaches but requiring its own, different assumptions. Most importantly, it is assumed that the only effect of antibiotic treatment on amino acid metabolism was to suppress the microbial supply. It is possible, however, that the effect of the antimicrobial therapy was 2-fold and that the Indian subjects had subclinical infections that were eliminated by the antibiotic treatment. Minor inflammation can increase leucine oxidation by 10% (21) and, if present, could have accounted for a substantial portion of the observed reduction in leucine oxidation after antimicrobial treatment. This would mean that the amino acid contribution from intestinal microbes was less than what was observed. However, the ESR and total white blood cell counts of the subjects did not suggest that there was any ongoing infection, although 4 of the subjects had ESR values that were at the upper end of the normal range. On the other hand, the change in microbial amino acid supply may have been underestimated if the gut microbiota were not effectively eliminated. There are differences in the susceptibility to antibiotics of different microbial species and in different geographical regions. Ciprofloxacin has been widely used in India in the recent past and it is possible that varying degrees of antibiotic resistance could be present in different bacteria in the gut, as has been observed for pathogenic bacteria (22). It is also possible that there could have been an increased intestinal loss of amino acids because of the effect of the antimicrobial therapy on intestinal permeability or absorption. However, intestinal permeability has not been shown to change after antibiotics in elective colorectal surgery patients (23), nor have any effects on amino acid absorption been reported for quinolones. It has been shown that some antibiotics, such as viriginiamycin, improve intestinal digestibility of proteins in pigs; however, it is difficult to make an unequivocal interpretation of these findings related to the present study (24). It is also unlikely that the antimicrobial treatment affected body leucine metabolism because the slope of the oxidation: intake response was similar before and after treatment.

In conclusion, it appears that the intestinal microbiota make a measurable contribution, of the order of 24–27% of the EAR, to the supply of leucine, and, by extension, of other indispensable amino acids to the body. Such a nutritionally relevant, microbially derived contribution to body amino acid homeostasis might partly explain how undernourished populations, living on cereal- based diets marginal in protein and amino acids, can nevertheless adapt and survive.

	ACKNOWLEDGMENTS

 
We thank Dr. T. Thomas for statistical analysis, Dr. M. Soares for discussions, and Dr. Paul Moughan for helpful discussions on the design of this experiment.

	FOOTNOTES

 
¹ Supported by a grant from the Department of Biotechnology, Government of India.

² Author disclosures: T. Raj, U. Dileep, M. Vaz, M. F. Fuller, and A. V. Kurpad, no conflicts of interest.

⁵ Abbreviations used: EAR, estimated average requirement; ESR, erythrocyte sedimentation rate; KIC, -ketoisocaproic acid.

Manuscript received 19 May 2008. Initial review completed 26 June 2008. Revision accepted 20 August 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

1. FAO/WHO/UNU Expert Consultation. Proteins and amino acid requirements in human nutrition. World Health Organ Tech Rep Ser. 2007;935:265.

2. FAO/WHO/UNU Expert Consultation. Energy and protein requirements. World Health Organ Tech Rep Ser. 1985;724:204.

3. Millward DJ, Jackson AA. Protein:energy ratios of current diets in developed and developing countries compared with a safe protein:energy ratio: implications for recommended protein and amino acid intakes. Public Health Nutr. 2004;7:387–405.[Medline]

4. Kurpad AV, Regan MM, Raj T, Vasudevan J, Kuriyan R, Gnanou J, Young VR. Lysine requirements of chronically undernourished adult Indian men, measured by a 24h indicator amino acid oxidation and balance technique. Am J Clin Nutr. 2003;77:101–8.[Abstract/Free Full Text]

5. Kurpad AV, Regan MM, Nazareth D, Nagaraj S, Gnanou J, Young VR. Intestinal parasites increase the dietary lysine requirement in chronically undernourished adult Indian men. Am J Clin Nutr. 2003;78:1145–51.[Abstract/Free Full Text]

6. Fuller MF, Garlick PJ. Human amino acid requirements: can the controversy be resolved? Annu Rev Nutr. 1994;14:217–41.[Medline]

7. Metges CC. Contribution of microbial amino acids to amino acid homeostasis of the host. J Nutr. 2000;130:S1857–64.[Abstract/Free Full Text]

8. Metges CC, El-Khoury AE, Henneman L, Petzke KJ, Grant I, Bedri S, Pereira PP, Ajami AM, Fuller MF, et al. Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects. Am J Physiol. 1999;277:E597–607.[Medline]

9. Torrallardona D, Harris CI, Fuller MF. Pigs' gastrointestinal microflora provide them with essential amino acids. J Nutr. 2003;133:1127–31.[Abstract/Free Full Text]

10. Torrallardona D, Harris CI, Fuller MF. Lysine synthesized by the gastrointestinal microflora of pigs is absorbed, mostly in the small intestine. Am J Physiol Endocrinol Metab. 2003;284:E1177–80.[Abstract/Free Full Text]

11. Vince A, O'Grady F, Dawson AM. The development of ileostomy flora. J Infect Dis. 1973;128:638–41.[Medline]

12. Kurpad AV, Regan MM, Raj T, Gnanou JV. Branched-chain amino acid requirements in healthy adult human subjects. J Nutr. 2006;136:S256–63.[Abstract/Free Full Text]

13. Durnin JV, Womersley J. Body fat assessed by total body density and its estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br J Nutr. 1974;32:77–97.[Medline]

14. Siri WE. Body composition from the fluid spaces and density: analysis of methods. In: Brozek J, Henschel A, editors. Techniques for measuring body composition. Washington, DC: National Academy of Sciences, National Research Council; 1961. p. 223–44.

15. Kurpad AV, Raj T, El-Khoury A, Kuriyan R, Maruthy K, Borgonha S, Chandukudlu D, Regan MM, Young VR. Daily requirement for and splanchnic uptake of leucine in healthy adult Indians. Am J Clin Nutr. 2001;74:747–55.[Abstract/Free Full Text]

16. Metges CC, Petzke KJ, El-Khoury AE, Henneman L, Grant I, Bedri S, Regan MM, Fuller MF, Young VR. Incorporation of urea and ammonia nitrogen into ileal and fecal microbial proteins and plasma free amino acids in normal men and ileostomates. Am J Clin Nutr. 1999;70:1046–58.[Abstract/Free Full Text]

17. Jackson AA, Gibson NR, Bundy R, Hounslow A, Millward DJ, Wootton SA. Transfer of (15)N from oral lactose-ureide to lysine in normal adults. Int J Food Sci Nutr. 2004;55:455–62.[Medline]

18. Millward DJ, Forrester T, Ah-Sing E, Yeboah N, Gibson N, Badaloo A, Boyne M, Reade M, Persaud C, et al. The transfer of ¹⁵N from urea to lysine in the human infant. Br J Nutr. 2000;83:505–12.[Medline]

19. Libao-Mercado AJ, Zhu CL, Cant JP, Lapierre HN, Thibault JN, Sève B, Fuller MF, de Lange CFM. Source of nitrogen for pig gut microbes: effect of feeding fermentable fiber. In: Ortigues-Marty I, editor. Energy and protein metabolism and nutrition: 2^nd International Symposium on Energy and Protein Metabolism and Nutrition; 2007 Sept 9–13; Vichy, France. Wageningen (The Netherlands): Wageningen Academic Publishers, EAAP publication No. 124; 2007. p. 415–16.

20. Libao-Mercado AJ, Zhu CL, Cant JP, Lapierre HN, Thibault JN, Sève B, Fuller MF, de Lange CFM. Dietary and endogenous amino acids are the main nitrogen sources for microbial protein synthesis in the upper gut of pigs. J Nutr. In press 2008.

21. Kurpad AV, Jahoor F, Borgonha S, Poulo S, Rekha S, Fjeld CR, Reeds PJ. A minimally invasive tracer protocol is effective in assessing the response of leucine kinetics and oxidation to vaccination in chronically energy-deficient adult males and children. J Nutr. 1999;129:1537–44.[Abstract/Free Full Text]

22. Taneja N. Changing epidemiology of Shigellosis and emergence of ciprofloxacin-resistant Shigellae in India. J Clin Microbiol. 2007;45:678–9.[Free Full Text]

23. Reddy BS, Macfie J, Gatt M, Larsen CN, Jensen SS, Leser TD. Randomized clinical trial of effect of synbiotics, neomycin and mechanical bowel preparation on intestinal barrier function in patients undergoing colectomy. Br J Surg. 2007;94:546–54.[Medline]

24. Decuypere JA, Dierick NA, Vervaeke IJ, Henderickx HK. Influence of virginiamycin on the digestive physiology in precaecal re-entrant cannulated pigs. Arch Tierernahr. 1991;41:373–93.[Medline]

This article has been cited by other articles:

				K. I. Bryant, R. N. Dilger, C. M. Parsons, and D. H. Baker Dietary L-Homoserine Spares Threonine in Chicks J. Nutr., July 1, 2009; 139(7): 1298 - 1302. [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 Raj, T. Articles by Kurpad, A. V. Search for Related Content PubMed PubMed Citation Articles by Raj, T. Articles by Kurpad, A. V.