1. Volume 140, Issue 4, Pages 1314-1321 (April 2011)
Inosine Triphosphate Protects Against Ribavirin-Induced Adenosine Triphosphate Loss by Adenylosuccinate Synthase Function 
Yuki Hitomi, Elizabeth T. Cirulli, Jacques Fellay, John G. McHutchison, Alexander J. Thompson, Curtis E. Gumbs, Kevin V. Shianna, Thomas J. Urban, David B. Goldstein 
Gastroenterology 
Volume 140, Issue 4, Pages (April 2011)
DOI: /j.gastro
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2. Figure 1
Localization and frequencies of functional human ITPA variants. (A) Localization of 2 human ITPA functional variants, which are known to reduce the activity of inosine triphosphatase. Rs is a nonsynonymous variant located in the second exon that substitutes threonine for phenylalanine at amino acid position 32. Rs is located in the second intron, and the minor allele results in altered splicing efficiency and reduced expression of ITPA. Orange bars indicate the coding region of ITPA, and blue bars indicate untranslated region (UTR). (B) Distribution of each ITPA genotype combination in a large cohort of healthy control samples (n = 1281).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
ITP is not used by human erythrocyte ATPase. Although ATP was hydrolyzed by human erythrocyte ATPase, ITP was not. Increased Pi was detected after a 30-minute incubation at 37°C. Ouabain was used as a Na+,K+-ATPase inhibitor. Plotted data represent averages ± standard error of mean of triplicated assays. Representative data from 3 independent experiments are shown. *P < .01, **P < .001 (Student t test).
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
ITP is used by human ADSS. (A) ITP can replace GTP when ADSS generates adenylosuccinate and Pi from IMP and aspartate. (B) The relationship of ITP with purine biosynthesis. ITP was not used by ATPase (Figure 2). ATP is phosphorylated from AMP via adenosine diphosphate (ADP). AMP is made from IMP and aspartate via adenylosuccinate by ADSS using GTP as an energy source. (C) ADSS was expressed in the human erythrocyte. Each number represents an independent individual. Human erythrocytes were lysed, and total cell lysates were subjected to Western blot analysis for human ADSS. (D and E) Recombinant human ADSS was made. Recombinant proteins before (GST-ADSS) and after (ADSS and GST) digestion of the GST tag by specific protease were subjected to Western blot analysis for human ADSS (D) and GST tag (E). (F) ITP could be used by human ADSS instead of GTP. A significant increase in adenylosuccinate was detected after 30-minute incubation at 25°C. Plotted data represent averages ± standard error of mean of triplicated assays. Representative data from 3 independent experiments are shown. *P < .05, **P < .01 (Student t test).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
RBV-induced erythrocyte ATP reduction is alleviated by the hemolysis protective ITPA genotype. (A–C) There are no differences in erythrocyte characteristics between ITPA genotypes before RBV stimulation. Hematocrit (Htc; A), mean corpuscular volume (MCV; B), and ATP quantity (C) were measured before stimulation with RBV. We made a comparison between 7 individuals with at least 2 minor alleles of functional ITPA variants, shown as “Protective” (including 2 homozygous for rs minor allele, 2 homozygous for rs minor allele, and 3 compound heterozygotes), and 8 without any minor alleles of functional ITPA variants shown as “Wild type.” (D) After 6 hours, 12 hours, and 24 hours of RBV stimulation (1 mmol/L), erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. However, alleviation of ATP reduction by the hemolysis protective ITPA genotype was canceled by ADSS inhibitor 6-mercaptopurine (6-MP) (100 μmol/L). Plotted data represent averages ± standard error of mean among individuals with each of the ITPA genotypes. Representative data from 3 independent experiments are shown. *P < .05, **P < .01 (Mann–Whitney U test).
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
There are no differences in hemolysis between ITPA genotypes with or without stimulation by RBV or 6-MP in vitro. (A) Linear range of the Hb enzyme-linked immunosorbent assay (ELISA). Hemolysis was measured by detection of Hb in the supernatant prior to lysis. Supernatants from treated cells were diluted to achieve Hb concentrations within the linear range of the Hb ELISA assay (range, 6.25–400 ng/mL). (B) Hemolysis was measured by Hb ELISA using supernatant after 24 hours of RBV stimulation. Plotted data represent averages ± standard error of mean among individuals with each of the ITPA genotypes. Representative data from 3 independent experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
A model for how accumulated ITP caused by human genetic ITPA variants protects from RBV-mediated hemolytic anemia. (A) Because wild-type ITPA hydrolyses ITP to IMP, ITP accumulation is not seen in erythrocytes. Therefore, erythrocytes show severe RBV-induced ATP reduction. (B) Accumulated ITP in erythrocytes with ITPA variants restores the activity of ADSS in the setting of GTP depletion. Therefore, RBV-induced ATP reduction is mitigated. Adenosine, inosine, and guanosine are incorporated from outside of the erythrocyte using es-nucleotide transporters (ENTs). NBMPR inhibits ENTs, which facilitate nucleoside uptake into the erythrocyte; 6-MP inhibits the pathways that require IMP; and RBV inhibits both ENT and inosine monophosphate dehydrogenase (IMPDH).
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Difference in RBV-induced ATP depletion by ITPA genotype occurs via ADSS. Although there was no difference in erythrocyte ATP levels among ITPA genotypes with IMP-AMP pathway inhibition by 6-MP (with only the adenosine salvage pathway existing), erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype with inhibition of the adenosine salvage pathway by NBMPR (with only IMP-AMP pathway existing) as well as with RBV stimulation. Erythrocytes (7 with the protective genotype and 8 with the wild-type genotype) were stimulated with RBV (1 mmol/L), 6-MP (100 μmol/L), or NBMPR (10 μmol/L) for 24 hours. Plotted data represent averages ± standard error of mean among individuals with each of the ITPA genotypes. Representative data from 3 independent experiments are shown. *P < .01 (Mann–Whitney U test).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions