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* Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, New Jersey, USA;
Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands;
Clinic for Special Children, Strasburg, Pennsylvania, USA;
Department of Medicine, UCLA Medical School, Los Angeles, California, USA; and
|| Institute of Bioorganic Chemistry, Polish Academy of Sciences, Pozna
, Poland
2Correspondence: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, 225 Warren St., Newark, NJ 07101-1709, USA. E-mail: jakubows{at}umdnj.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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38%) by urinary excretion. In mice, hyperhomocysteinemia secondary to a high-methionine diet leads to 3.7-fold and 25-fold increases in plasma and urinary Hcy-thiolactone levels, respectively. Thus, we conclude that hyperhomocysteinemia leads to significant increases in the atherogenic metabolite Hcy-thiolactone in humans and mice.—Chwatko, G., Boers, G. H. J., Strauss, K. A., Shih, D. M., Jakubowski, H. Mutations in methylenetetrahydrofolate reductase or cystathionine ß-syntase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice.
Key Words: genetic hyperhomocysteinemia • dietary hyperhomocysteinemia • atherosclerosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Although severe hyperhomocysteinemia is rare, mild hyperhomocysteinemia is quite prevalent in a general population and is associated with an increased risk of cardiovascular (2)
and neurodegenerative diseases such as Alzheimer’s (3)
. Perhaps the strongest evidence that Hcy may play a causal role in cardiovascular disease is the favorable effect of Hcy lowering in patients with cystathionine ß-synthase (CBS) deficiency (4)
. Small trials in humans (5)
and studies of dietary (induced by a high-Met diet) or genetic hyperhomocysteinemia in animal models (1)
suggest that Hcy may also play a causal role in cardiovascular disease in general populations. Although large clinical trials testing whether lowering mild elevations of Hcy can lead to better vascular outcomes have been controversial (5)
, high-risk stroke patients do benefit from lowering of plasma Hcy by vitamin supplementation (6
, 7)
.
Although it is a normal metabolite, Hcy excess is extremely toxic to cultured cells (8
9
10)
, including human (11
12
13)
, animal (14)
, yeast (15
, 16)
, and bacterial (17)
. Why Hcy is toxic is not entirely clear and is a subject of intense study. In all organisms, including humans, a small fraction of total Hcy is metabolized to Hcy-thiolactone by methionyl-tRNA synthetase (Met-RS) in an error-editing reaction in protein biosynthesis when Hcy becomes mistakenly selected in place of Met (reviewed in refs. 8
9
10
). One hypothesis suggests that the conversion to Hcy-thiolactone contributes to Hcy toxicity and is involved in atherosclerosis in humans (8
9
10
, 18
, 19)
. Indeed, Hcy-thiolactone appears to be more toxic to human cells than Hcy itself (reviewed in ref. 10
).
Previous human tissue culture studies have shown that the flow through the Hcy-thiolactone pathway is increased when Hcy metabolism is impaired by mutations in the CBS gene (18)
or by limiting the supply of folate (18
, 19)
. However, it was not known whether genetic disorders in Hcy metabolism, or a high-Met diet, affect Hcy-thiolactone levels in mammals. The present work shows that Hcy-thiolactone levels are elevated in human CBS- and methyleneterahydrofolate (MTHFR) -deficient patients. We also show that Hcy-thiolactone levels are elevated in hyperhomocysteinemic mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Eleven of these patients were 833T > C (Ile278Thr) homozygotes, two were compound heterozygotes for the 833T > C and 1111G > A (Val371Met) mutations, and one was a compound heterozygote for the 373C > T (Arg125Trp) and 1301C > A (Thr434Asn) mutations. CBS activity in fibroblasts derived from these patients was < 2.5% of control CBS activity from unaffected individuals (20)
. Patients were initially diagnosed at ages 2 to 54 on the basis of clinical manifestations of CBS deficiency (ectopia lensis, Marfanoid appearance, osteoporosis), in combination with a quantitative determination of severe hyperhomocysteinemia and hypermethioninemia. Five CBS-deficient patients survived a vascular event before diagnosis. All patients were on Hcy-lowering treatment (vitamin B6) following diagnosis. Blood samples were taken for the preparation of plasma as described previously (21
, 22)
.
MTHFR-deficient human subjects
MTHFR-deficient subjects were from the Amish population centered around Somerset County, PA, USA. Four patients had hyperhomocysteinemia due to homozygous 1129C > T (Arg377Cys) missense mutation in the MTHFR gene (23)
diagnosed at age 18 years, 4 years, 15 months, or 1 wk. The 1129C > T mutation was detected by DNA sequencing of the entire coding region of the MTHFR gene or by the polymerase chain reaction (PCR) (23)
. After diagnosis, the patients were on Hcy-lowering therapy (betaine). Six unaffected siblings or parents, heterozygous for the 1129C > T MTHFR mutation, were also studied. Nine unrelated healthy adult subjects with normal plasma tHcy levels were used as controls (Table 1
).
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Mice
Two-month-old mice (strain C57BL/6J; Jackson laboratory, Bar Harbor, ME, USA) were fed a control diet containing 0.5% Met, a high-Met isocaloric diet containing 1.5% Met (TD.04352 or TD.04353, respectively, Harlan Teklad, Madison, WI, USA), or a high-Hcy diet that consisted of TD.04352 plus 0.09% D,L-Hcy in drinking water for 6 wk.
Principles of the Hcy-thiolactone assay
Special physical chemical properties (8)
were exploited to achieve selective extraction of Hcy-thiolactone and to remove most of the Hcy and other interfering compounds from plasma or urine samples as described previously (21
, 22)
. The 
-amino group of Hcy-thiolactone has a pKa = 6.67 (Eq. 1
) (24)
, which is much lower than the pKa of 9.5 characteristic of -amino groups of amino acids. The exceptionally low pKa makes Hcy-thiolactone essentially neutral at pH > 7.4 and mostly positively charged at a pH near 6 (Eq. 1
); ionization of other amino acids, except histidine, essentially is not affected by changing pH from 6 to 8. Thus, we first adjusted the samples to pH = 8.0 to convert Hcy-thiolactone to a neutral hydrophobic form. The hydrophobicity of the neutral form of Hcy-thiolactone is sufficient to facilitate its transfer to the organic phase upon extraction with chloroform/methanol (21)
or its binding to charcoal (22)
. Conversion of the neutral form to a positively charged form upon acidification with diluted HCl facilitates re-extraction of Hcy-thiolactone from the organic phase (21)
or release from charcoal (22)
. The pH-dependent changes in the ionization status of Hcy-thiolactone (Eq. 1
) also allow its selective separation from Hcy and other amino acids on a polysulfoethyl aspartamide HPLC column (21
, 22)
.
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| (1) |
Sample preparation for Hcy-thiolactone assays in plasma
EDTA plasma samples were prepared as described previously (21
, 22)
. Human or mouse EDTA plasma samples (0.4 ml) were deproteinized by ultrafiltration through Millipore 10 kDa cutoff membranes at 4°C. The protein-free ultrafiltrate (0.2 ml) was adjusted to pH 8.0 with 0.01 ml of 1 M K2HPO4, and Hcy-thiolactone was extracted with 1 ml chloroform/methanol (2:1 by volume) mixture at room temperature. Hcy-thiolactone was re-extracted from the organic phase with 0.2 ml of 0.1 M HCl. Recovery of the extraction was 61.8 ± 7.7% (21
, 22)
. The aqueous phase was lyophilized on a Labconco concentrator, the residue was dissolved in 20 µl of deionized water, and 10 µl aliquots were mixed with 10 µl mobile phase and analyzed by cation exchange HPLC.
Sample preparation for Hcy-thiolactone assays in urine
Mouse urine (0.5 ml) was ultrafiltered through a 10 kDa cutoff membrane (Ultrafree 10 kDa Biomax, Millipore, Billerica, MA, USA) at 4°C. The pH of the ultrafiltrate (0.25 ml) was adjusted to 8.0 by the addition of 1M K2HPO4 (20 µl) to convert the positively charged form of Hcy-thiolactone to a neutral form (Eq. 1
, ref. 22
), which was then adsorbed on 10 mg PBS-washed activated charcoal (Sigma-Aldrich, St. Louis, MO, USA; cat. no. C 7606). The charcoal was washed with 1 M NaCl (twice with 0.1 ml each time), 0.1 ml PBS, and 0.1 ml deionized water. Conversion of the adsorbed Hcy-thiolactone to the positively charged form by acidification with 10 mM HCl (three times with 0.1 ml each time) led to release of Hcy-thiolactone from the charcoal. The acid eluates were lyophilized, dissolved in 100 µl mobile phase, and 10 µl aliquots were subjected to cation exchange HPLC.
Sample preparation for tHcy assays in plasma or urine
Human or mouse plasma tHcy was determined as described (16
, 21)
by using a procedure that involves conversion to Hcy-thiolactone, which is then quantified by HPLC. Mouse urinary tHcy was determined by a similar procedure. Urine (200 µl) was treated with 4 µl 0.5 M dithiothreitol and ultrafiltered through a Millipore 10 kDa membrane at 4°C. A 100 µl portion of the ultrafiltrate was treated with 2 µl of 1 M dithiothreitol and 10 µl 12 M HCl for 30 min at 100°C. A 10 µl aliquot of the treated ultrafiltrate was lyophilized and dissolved in 200 µl mobile phase; 20 µl of this solution was applied to a cation exchange HPLC column.
HPLC analyses
Hcy-thiolactone was analyzed by a cation exchange HPLC with postcolumn derivatization and fluorescence detection as described previously (21
, 22)
. Beckman-Coulter System Gold Nouveau HPLC instrumentation containing multimode 508 autosampler, advanced gradient solvent delivery module 126, and a Jasco 1520 fluorescence detector was used. A manual injector (7725i rheodyne) with an 0.1 ml loop was used to analyze single samples. Chromatograms were analyzed by Gold Nouveau chromatography workstation software for Windows.
Samples (10–20 µl) were injected into a cation exchange PolySULFOETHYL Aspartamide column [150x1 mm (i.d.), 5 µm bead size, 300 Å pore size; Poly LC, Inc., Columbia, MD, USA]. The column was eluted isocratically with 10 mM sodium phosphate buffer, pH 6.6, containing 5 mM NaCl at a flow rate of 0.15 ml/min. Under these conditions Hcy-thiolactone is retained on the column and elutes at 8 min, whereas Hcy elutes in a void volume at 3 min (21
, 22)
.
For postcolumn derivatization and detection, the effluent was mixed in a 3-way T-valve with 2.5 mM OPA in 0.25 M NaOH, delivered at a flow rate of 0.07 ml/min. The mixture passed through a TeflonTM tubing reaction coil [0.3 mm (i.d.) x 3 m], then monitored with a Jasco 1520 fluorescence detector using excitation at 370 nm and fluorescence emission at 480 nm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Because MTHFR-deficient patients were on Hcy-lowering therapy (see Materials and Methods), the Hcy-thiolactone concentrations represent minimal values. In one patient for whom samples were obtained before Hcy-lowering therapy, the therapy resulted in a lowering of plasma Hcy-thiolactone from 47.3 ± 1.7 nM (two assays on a single sample) to 16.6 ± 8.5 nM (assays on three samples) (tHcy was lowered from 208.0 µM before therapy to 66.2 µM after therapy). Plasma Hcy-thiolactone levels in the MTHFR homozygous patients were significantly higher than in heterozygous (0.5±0.29 nM, P=0.02) or wild-type individuals (0.2±0.14 nM, P=0.03). Significantly more plasma Hcy-thiolactone was present in heterozygous than in wild-type subjects (0.5±0.29 vs. 0.2±0.14 nM, P=0.006).
Plasma tHcy concentrations in the homozygous MTHFR-deficient patients were elevated, as expected, and varied from 22.9 to 68.0 µM with a mean value of 50.1 ± 15.1 µM and a median of 44.9 µM (Table 1)
. Mean plasma tHcy concentrations in heterozygous MTHFR subjects and wild-type individuals were in the normal range: 7.8 ± 2.8 µM and 7.2 ± 0.9 µM, respectively. There was a significant linear relationship between plasma tHcy/Met ratios and plasma Hcy-thiolactone (Fig. 1
A).
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Hcy-thiolactone levels in CBS-deficient human subjects
Plasma concentrations of Hcy-thiolactone in the CBS-deficient patients studied here were elevated with a mean value of 14.4 ± 30.4 nM and a median of 2.8 nM (range<0.1 to 100 nM) (Table 1)
, similar to corresponding values in the MTHFR-deficient patients. Because CBS-deficient patients, like the MTHFR-deficient patients, were on Hcy-lowering therapy (see Materials and Methods), these Hcy-thiolactone concentrations represent minimal values. Plasma concentrations of tHcy in the CBS-deficient patients were also elevated, as expected, with a mean value of 36.1 ± 25.8 µM and a median of 25.5 µM (Table 1)
. There was a relatively weak but significant relationship between plasma tHcy/Met ratios and plasma Hcy-thiolactone in a group of CBS-deficient patients (Fig. 1B
).
Hcy-thiolactone and tHcy levels in the mouse
Plasma Hcy-thiolactone levels in mice fed a normal diet had a mean value of 3.7 ± 2.1 nM (Table 2
), higher than Hcy-thiolactone levels in normal human plasma (Table 1)
. Urinary concentrations of Hcy-thiolactone in mice were 140 ± 220 nM, 38-fold higher than those in plasma (Table 2)
and similar to Hcy-thiolactone levels in human urine (22)
. Mouse plasma tHcy levels were 3.0 ± 1.5 µM, 2.4-fold lower than tHcy levels in normal human plasma (Table 1)
. Mouse urinary tHcy levels were 45 ± 14 µM, 15-fold higher than those in mouse plasma and 18-fold or 5.3-fold higher than tHcy levels in human (22)
or rat (25)
urine, respectively.
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In mice fed a high-Met diet, Hcy-thiolactone concentrations increased 3.7-fold (to 13.8±4.8 nM) in plasma and 25-fold (to 3490±3780 nM) in urine. Plasma and urinary tHcy increased 17.3-fold (to 51.8±22.7 µM) and 30-fold (to 1360±840 µM), respectively (Table 2)
.
In mice fed a high-Hcy diet, plasma Hcy-thiolactone did not change significantly whereas urinary Hcy-thiolactone increased 3.6-fold, to 496 ± 151 nM. Plasma and urinary tHcy increased 7.1-fold (to 21.4±10.2 µM) and 7.5-fold (to 338±146 µM), respectively (Table 2)
.
In mice fed a high-Met or high-Hcy diet, relative urinary concentrations of Hcy-thiolactone were significantly higher (urinary HTL/plasma HTL=252 or 155) than in mice fed a normal diet (urinary HTL/plasma HTL=37, P<0.05) (Table 2)
. In contrast, relative urinary concentrations of tHcy in mice were hardly affected by the diet (last column in Table 2
).
To exclude the possibility that the increases in Hcy-thiolactone seen in mice fed hyperhomocysteinemic diets are not due to contamination, we assayed commercial preparations of L-methionine and D,L-Hcy (from Sigma-Aldrich) for Hcy-thiolactone. We found no Hcy-thiolactone in L-methionine (<10–5 mol/mol). D,L-Hcy contained 5.7 ± 1.1 x 10–5 mol Hcy-thiolactone/mol Hcy, but this did not account for the Hcy-thiolactone levels observed in mice fed a high-Hcy diet (Table 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present work also demonstrates that insights into Hcy-thiolactone metabolism obtained in our previous ex vivo studies with cultured human and rodent cells are valid for the human body. For example, we previously showed that cultured human CBS-deficient fibroblasts synthesize more Hcy-thiolactone and Hcy than normal fibroblasts (18)
, and the present work shows a similar effect of CBS deficiency on Hcy-thiolactone levels in the human body (Table 1)
. Similarly, our earlier work has shown that limiting the availability of folic acid greatly enhances Hcy-thiolactone synthesis in human fibroblasts (18)
and vascular endothelial cells (19)
, whereas the present work shows a similar effect of 5-methyltetrahydofolate deficiency (caused by the MTHFR mutation) on Hcy-thiolactone synthesis in human subjects (Table 1)
. Our previous in vitro and ex vivo tissue culture work has demonstrated that Hcy-thiolactone is a product of Hcy editing by Met-RS (8
9
10)
. Linear relationships between plasma tHcy/Met ratios and Hcy-thiolactone in MTHFR- and CBS-deficient subjects observed in the present work (Fig. 1A, B
) suggest that Met-RS is a major enzyme involved in Hcy-thiolactone synthesis in the human body.
Oral administration of Met or Hcy is often used as a useful model of experimental hyperhomocysteinemia (1)
. We found that a high-Met diet also causes 3.7-fold and 25-fold increases in plasma and urinary Hcy-thiolactone, respectively. A high-Hcy diet was somewhat less effective than a high-Met diet in increasing plasma or urinary Hcy-thiolactone and tHcy levels in mice (Table 2)
, although the measured intake of water did not change due to the presence of Hcy. We also found that the distribution of Hcy-thiolactone between plasma and urine is similar in mice fed a normal diet and in humans; much higher (37-fold for mice, Table 2
, and 100-fold for humans, ref. 22
) Hcy-thiolactone concentrations accumulate in urine than in plasma, suggesting similar urinary clearances of Hcy-thiolactone in mice and humans. In humans, >95% of the filtered Hcy-thiolactone is excreted in the urine (22)
. In addition, urinary clearance of Hcy-thiolactone in mice appears to be much more effective under the conditions of hyperhomocysteinemia, as suggested by significantly higher urinary/plasma Hcy-thiolactone ratios in mice fed hyperhomocysteinemic diets than in mice fed a normal diet (Table 2)
.
Because excess Met prevents access of Hcy to the active site of Met-RS and the formation of Hcy-thiolactone (9
, 19)
, the increased accumulation of Hcy-thiolactone in mice on a high-Met diet is surprising. One possible explanation for the enhanced accumulation of Hcy-thiolactone in mice is that, in addition to Met-RS, other enzymes that are not inhibited by Met, such as Ile- and/or Leu-RS (9)
, contribute to Hcy-thiolactone synthesis in the mouse. Human Ile- and Leu-RS (H. Jakubowski, unpublished data), like their bacterial counterparts (26)
, have the ability to catalyze the synthesis of Hcy-thiolactone.
In contrast to humans, mice have much higher relative urinary tHcy: in mice, the urinary/plasma tHcy ratio is 15 (Table 2)
whereas in humans the ratio is 0.4 (22)
. In rats the urinary/plasma tHcy ratio is 1.9 (25)
, somewhat higher than in humans but not as high as in mice. A higher urinary/plasma tHcy ratio may reflect a higher proportion of free Hcy (not bound to plasma proteins) in mouse plasma. However, to our best knowledge the free/tHcy ratio in the mouse has not been published, whereas in rats the free/tHcy ratio is 0.42 (ref. 27
), somewhat higher than in humans (0.30, ref. 28
). The high urinary/plasma tHcy ratio in the mouse suggests that a much greater amount of plasma tHcy is eliminated by the kidney in mice than in humans. In humans, 99% of filtered Hcy is reabsorbed and only 1% is excreted (22
, 28)
. Assuming that the urinary/plasma tHcy ratio reflects tHcy clearance, one can calculate that a significant amount of plasma tHcy (38%) is eliminated by the kidney in the mouse. Significant urinary elimination of tHcy in the mouse suggests that mice may be more resistant to Hcy toxicity than humans, and therefore may not be an ideal model for human hyperhomocysteinemia. The mechanism underlying different distributions of tHcy in mice and humans warrants further investigation.
Hyperhomocysteinemia secondary to genetic deficiencies in the CBS or MTHFR gene or to a high-Met diet is known to cause atherosclerosis in humans and mice (1
, 4)
. In animal models, treatment with Hcy-thiolactone causes pathophysiological changes similar to those observed in severe hyperhomocysteinemia. For example, infusions with Hcy-thiolactone or an Hcy-thioactone-supplemented diet produce atherosclerotic changes in baboons (29)
or rats (30)
whereas treatments with Hcy-thiolactone causes developmental abnormalities in eyes of chick embryos, including optic lens dislocation (31)
characteristic of CBS-deficient human patients (4
, 20)
.
Our present findings that Hcy-thiolactone levels are significantly elevated in hyperhomocysteinemic humans and mice provide support to a hypothesis that the metabolic conversion of Hcy into Hcy-thiolactone contributes to the pathophysiology of hyperhomocysteinemia (Fig. 2
). According to this hypothesis, Hcy-thiolactone is a toxic metabolite because it causes protein N-homocysteinylation through the formation of amide bonds with 
-amino groups of protein lysine residues (8
9
10
, 18
, 19
, 32
33
34)
. Previous studies have shown that hyperhomocysteinemia leads to increases in N-Hcy-protein levels in humans (33
34
35
36)
. N-Hcy-proteins are detrimental to the human body and contribute to two important aspects of pathophysiology: immune activation and thrombogenesis (Fig. 2)
. For example, N-Hcy-proteins formed in the human body (33
, 34)
induce anti-N-Hcy-protein autoantibodies whose levels are associated with stroke and coronary artery disease (37
38
39
40)
. N-homocysteinylation of fibrinogen by Hcy-thiolactone (32)
, which occurs in the human body (33)
, affects fibrin clot permeability and susceptibility to lysis in humans, causing increased thrombogenesis (41
, 42)
.
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In conclusion, our present data demonstrate that hyperhomocysteinemia secondary to genetic deficiencies in Hcy metabolism or induced by a diet leads to significant increases in the atherogenic metabolite Hcy-thiolactone in humans and mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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, 90-236 Lód, Poland. 
Received for publication October 12, 2006. Accepted for publication January 4, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ski, M., Trzeciak, W., Kamierski, R., Jakubowski, H. (2004) Autoantibodies against N-homocysteinylated proteins in humans: implications for atherosclerosis. Stroke 35,1299-1304This article has been cited by other articles:
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  N. Wu, Y. L. Siow, and K. O Induction of hepatic cyclooxygenase-2 by hyperhomocysteinemia via nuclear factor-{kappa}B activation Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2009; 297(4): R1086 - R1094. [Abstract] [Full Text] [PDF]
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 M. Sikora and H. Jakubowski Homocysteine editing and growth inhibition in Escherichia coli Microbiology, June 1, 2009; 155(6): 1858 - 1865. [Abstract] [Full Text] [PDF]
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 H. Jakubowski, J. Perla-Kajan, R. H. Finnell, R. M. Cabrera, H. Wang, S. Gupta, W. D. Kruger, J. P. Kraus, and D. M. Shih Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice FASEB J, June 1, 2009; 23(6): 1721 - 1727. [Abstract] [Full Text] [PDF]
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H. Jakubowski, G. H. J. Boers, and K. A. Strauss Mutations in cystathionine {beta}-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans FASEB J, December 1, 2008; 22(12): 4071 - 4076. [Abstract] [Full Text] [PDF]
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 S. H. Schirmer, J. O. Fledderus, P. T.G. Bot, P. D. Moerland, I. E. Hoefer, J. Baan Jr, J. P.S. Henriques, R. J. van der Schaaf, M. M. Vis, A. J.G. Horrevoets, et al. Interferon-{beta} Signaling Is Enhanced in Patients With Insufficient Coronary Collateral Artery Development and Inhibits Arteriogenesis in Mice Circ. Res., May 23, 2008; 102(10): 1286 - 1294. [Abstract] [Full Text] [PDF]
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