Auxotrophy-Based Detection of Hyperornithinemia in Mouse Blood and Urine

Gyrate atrophy of the choroid and retina (GACR) is a hereditary form of progressive blindness caused by homozygosity for loss-of-function mutations in the ornithine aminotransferase gene (Oat). The high levels of circulating ornithine that lead to ophthalmic symptoms in young adults are also displayed by 2 ornithine aminotransferase (OAT)-deficient mouse models of GACR. Here, we have developed an inexpensive and quantitative bacteria-based test for detecting hyperornithinemia in blood or urine samples from these mutant mice, a test that we suggest could be used to facilitate the identification and treatment of OAT-deficient humans before the onset of visual impairment.

Keywords
argE mutant E coli; gyrate atrophy of the choroid and retina; ornithine biosensor; metabolic screening; animal models of human disease

Authorship

Kenneth M. Palanza

Biomolecular Sciences, Central Connecticut State University, New Britain, CT, USACentral Connecticut State UniversityUSANew Britain, CT, USABiomolecular Sciences, Central Connecticut State University, New Britain, CT, USA

Alex V. Nesta

Renukanandan Tumu

Cherie M. Walton

Michael A. Davis

Thomas R. King Corresponding Author: Thomas R. King, PhD, Biomolecular Sciences, Central Connecticut State University, 1615 Stanley Street, New Britain, CT 06053, USA. Email: kingt@ccsu.edu

Corresponding Author: Thomas R. King, PhD, Biomolecular Sciences, Central Connecticut State University, 1615 Stanley Street, New Britain, CT 06053, USA. Email: kingt@ccsu.edu

Authors’ Note

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Declaration of Conflicting Interests

The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Figures

Figures (6)

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Figure 1
Pathway of arginine biosynthesis in mammals. Mutation of Oat (which encodes ornithine aminotransferase) blocks the synthesis of ornithine from pyrroline-5-carboxylate (P-5-C) in neonates, which is required for the synthesis of arginine in the urea cycle. In neonatal mice (but not humans), ornithine aminotransferase (OAT) deficiency leads to blood levels of arginine that are insufficient to support normal growth. In adult mice and humans, OAT deficiency blocks the conversion of ornithine to P-5-C, leading to an accumulation of ornithine in the blood that, over time, causes progressive retinal degeneration.

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Figure 2
Pathway of arginine biosynthesis in Escherichia coli. Mutationof argE (which codes for acetylornithine deacetylase) prohibits thesynthesis of ornithine, which is required for the synthesis of arginine.Growth of argE auxotrophs on minimal media can be rescued by supplementation with ornithine or other downstream intermediates.

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Figure 3
Rescue of the auxotrophic, argE mutant Escherichia coli strain CAG12185 on M9 minimal media by addition of exogenous ornithine or physiological fluids from mouse mutants with hyperornithinemia. A to D, Culture dishes contained M9 minimal lactose media with a top agar layer containing bacterial strain E coli CAG12185 and X-Gal. Sterile paper discs containing 30 µL of 5 mmol/L ornithine (resulting in 1.5 µg ornithine in the disc), 2.5 mmol/L (0.75 µg), 1.25 mmol/L (0.38 µg), 0.63 mmol/L (0.19 µg), or 0.31 mmol/L (0.09 µg) ornithine were placed on the top agar in panel A, as labeled. Ornithine diffusing from the disc in sufficient quantities can rescue the growth of auxotrophic bacteria contained within the top agar, and hydrolysis of X-Gal produces a nondiffusing blue pigment that reports the presence of accumulating bacteria. The most bacterial growth can be seen around the disc containing the 1.5 µg ornithine, which shows a more intensely colored and larger blue halo. Bacterial growth decreases with ornithine concentration until no growth can be observed around the discs containing 0.19 µg or less ornithine. In panels B to D, paper discs were loaded instead with 30 µL of: +/+. +/rhg. +/Oat^∆. rhg/rhg, and rhg/Oat^∆ urine, plasma, or serum as indicated. Growth is observed around the discs containing fluids from mutant (rhg/rhg and rhg/Oat^∆) mice but not around discs containing fluids from mice with at least 1 wild-type allele of Oat. E to H, E coli CAG12185 was cultured in microtiter wells that contained M9 minimal glucose media and wild-type urine (panel E) or serum (panel F) that had been spiked with various concentrations of ornithine, as indicated. In both of these “standard” assays, rescue of bacterial growth is, again, proportional to the concentration of ornithine supplied, although bacterial growth appears to be stimulated by the presence of serum, even with no ornithine added, which never stimulated growth in cultures that included urine instead of serum. In panels G and H, E coli CAG12185 was cultured in microtiter wells that contained M9 minimal glucose media and urine or serum, respectively, from mice with the 5 genotypes indicated. In both “experimental” assays, rhg/Oat^∆ urine or serum samples rescued more growth than rhg/rhg samples, but although urine from +/+. +/rhg, or +/Oat^∆ mice rescued no bacterial growth in these cultures, serum samples from wild-type mice with these genotypes stimulated modest increases in turbidity.

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Figure 4
Bradford assay performed on mouse urine or serum to determine soluble protein concentration. A and B, Standard curves relating known protein concentration [bovine serum albumin (BSA) dissolved in wild-type mouse urine (panel A) or serum (panel B)] to absorbance at 595 nm. C, Assay performed in parallel with the standard (A) on 10-fold diluted urine samples from the 5 genotypes of mice, as indicated. All assays were performed in triplicate. Using the equation from the standard curve, the protein concentration could be determined in the overall urine sample. The range of protein concentration in urine was 4.0 to 5.7 µg/µL (an average of 4.7 ± 0.8 µg/µL). D, Assay performed in parallel with the standard (B) on 200-fold diluted serum samples from the 5 genotypes of mice, as indicated. The range of protein concentration in serum was 85.8 to 99.2 µg/µL (an average of 91.3 ± 3.6 µg/µL).

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Figure 5
Standard curves relating the growth of Escherichia coli strain CAG12185 to the known concentration of ornithine added to cultures containing wild-type mouse urine (panel A) or serum (panel B). These standard curves correspond to the standard assays shown in Figure 3, panels E and F. Turbidity in the culture wells at 48 hours of incubation (indicative of bacterial growth and based on the average of 3 measures of absorbance at 600 nm) was plotted against the known concentrations of ornithine present in the microtiter well. A 3-parameter sigmoidal logistic regression was generated from the plotted values to produce an equation relating ornithine concentration to growth. From the equation and the absorbance readings resulting from bacterial growth in the physiological fluid samples gathered from mutant rhg/rhg or rhg/Oat^∆ mice, ornithine concentrations in the respective physiological fluid samples were determined (see text).

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Figura 6
Rescue of the auxotrophic, argE mutant Escherichia coli strain BW6165 on M9 minimal media by addition of exogenous ornithine or physiological fluids from mouse mutants with hyperornithinemia. A and B, E coli BW6165 was cultured in microtiter wells that contained M9 minimal glucose media and wild-type urine (panel A) or serum (panel B) that had been spiked with various concentrations of ornithine, as indicated. In both standard assays, rescue of bacterial growth was proportional to the concentration of ornithine supplied. In panels C and D, E coli BW6165 was cultured in microtiter wells that contained M9 minimal glucose media and urine or serum, respectively, from mice with the 5 genotypes indicated. In both experimental assays, rhg/Oat^∆ urine or serum samples rescued more growth than rhg/rhg samples, but although urine from +/+. +/rhg, or +/Oat^∆ mice rescued no bacterial growth in these cultures, serum samples from wild-type mice with these genotypes sometimes stimulated modest increases in turbidity (eg, see +/rhg culture in panel D). Similar results were obtained in microtiter culture experiments that used E coli strain CAG12185 (see Figure 3). E and F, Standard curves relating the growth of E coli strain BW6165 to known concentrations of ornithine added in wild-type mouse urine (panel E) or serum (panel F), based on the average of 3 turbidity readings taken at 48 hours of incubation in the same assays shown in panels A and B, respectively. A 3-parameter sigmoidal logistic regression was generated from the plotted values to produce an equation relating ornithine concentration to growth. With this equation, bacterial growth observed in the experimental assays of mutant (rhg/rhg or rhg/Oat^∆) urine (panel C) or serum (panel D) can be interpreted as ornithine concentrations in the respective physiological fluid samples. From the standard curve shown in panel E, the ornithine concentration in urine from mutant rhg/rhg mice was 3.4 ± 0.2 mmol/L (N = 3) and from rhg/Oat^∆ mice was 4.7 ± 0.4 mmol/L (N = 3), in close agreement with values determined using E coli strain CAG12185 (see text). From the standard curve shown in panel F, the ornithine concentrations in mutant serum samples were found to be 1015 ± 35 µmol/L for rhg/rhg (N = 3) and 1139 ± 69 µmol/L for rhg/Oat^∆ serum (N = 3), again in good agreement with values determined using E coli CAG12185 and with values measured directly by Bisaillon et al¹⁰10 Bisaillon, JJ, Radden, LA II, Szabo, ET. The retarded hair growth (rhg) mutation in mice is an allele of ornithine aminotransferase (Oat). Mol Genet Metab Rep. 2014;1:378–390. (see text).

Figure 1 Pathway of arginine biosynthesis in mammals. Mutation of Oat (which encodes ornithine aminotransferase) blocks the synthesis of ornithine from pyrroline-5-carboxylate (P-5-C) in neonates, which is required for the synthesis of arginine in the urea cycle. In neonatal mice (but not humans), ornithine aminotransferase (OAT) deficiency leads to blood levels of arginine that are insufficient to support normal growth. In adult mice and humans, OAT deficiency blocks the conversion of ornithine to P-5-C, leading to an accumulation of ornithine in the blood that, over time, causes progressive retinal degeneration.

Figure 2 Pathway of arginine biosynthesis in Escherichia coli. Mutationof argE (which codes for acetylornithine deacetylase) prohibits thesynthesis of ornithine, which is required for the synthesis of arginine.Growth of argE auxotrophs on minimal media can be rescued by supplementation with ornithine or other downstream intermediates.

Figure 3 Rescue of the auxotrophic, argE mutant Escherichia coli strain CAG12185 on M9 minimal media by addition of exogenous ornithine or physiological fluids from mouse mutants with hyperornithinemia. A to D, Culture dishes contained M9 minimal lactose media with a top agar layer containing bacterial strain E coli CAG12185 and X-Gal. Sterile paper discs containing 30 µL of 5 mmol/L ornithine (resulting in 1.5 µg ornithine in the disc), 2.5 mmol/L (0.75 µg), 1.25 mmol/L (0.38 µg), 0.63 mmol/L (0.19 µg), or 0.31 mmol/L (0.09 µg) ornithine were placed on the top agar in panel A, as labeled. Ornithine diffusing from the disc in sufficient quantities can rescue the growth of auxotrophic bacteria contained within the top agar, and hydrolysis of X-Gal produces a nondiffusing blue pigment that reports the presence of accumulating bacteria. The most bacterial growth can be seen around the disc containing the 1.5 µg ornithine, which shows a more intensely colored and larger blue halo. Bacterial growth decreases with ornithine concentration until no growth can be observed around the discs containing 0.19 µg or less ornithine. In panels B to D, paper discs were loaded instead with 30 µL of: +/+. +/rhg. +/Oat^∆. rhg/rhg, and rhg/Oat^∆ urine, plasma, or serum as indicated. Growth is observed around the discs containing fluids from mutant (rhg/rhg and rhg/Oat^∆) mice but not around discs containing fluids from mice with at least 1 wild-type allele of Oat. E to H, E coli CAG12185 was cultured in microtiter wells that contained M9 minimal glucose media and wild-type urine (panel E) or serum (panel F) that had been spiked with various concentrations of ornithine, as indicated. In both of these “standard” assays, rescue of bacterial growth is, again, proportional to the concentration of ornithine supplied, although bacterial growth appears to be stimulated by the presence of serum, even with no ornithine added, which never stimulated growth in cultures that included urine instead of serum. In panels G and H, E coli CAG12185 was cultured in microtiter wells that contained M9 minimal glucose media and urine or serum, respectively, from mice with the 5 genotypes indicated. In both “experimental” assays, rhg/Oat^∆ urine or serum samples rescued more growth than rhg/rhg samples, but although urine from +/+. +/rhg, or +/Oat^∆ mice rescued no bacterial growth in these cultures, serum samples from wild-type mice with these genotypes stimulated modest increases in turbidity.

Figure 4 Bradford assay performed on mouse urine or serum to determine soluble protein concentration. A and B, Standard curves relating known protein concentration [bovine serum albumin (BSA) dissolved in wild-type mouse urine (panel A) or serum (panel B)] to absorbance at 595 nm. C, Assay performed in parallel with the standard (A) on 10-fold diluted urine samples from the 5 genotypes of mice, as indicated. All assays were performed in triplicate. Using the equation from the standard curve, the protein concentration could be determined in the overall urine sample. The range of protein concentration in urine was 4.0 to 5.7 µg/µL (an average of 4.7 ± 0.8 µg/µL). D, Assay performed in parallel with the standard (B) on 200-fold diluted serum samples from the 5 genotypes of mice, as indicated. The range of protein concentration in serum was 85.8 to 99.2 µg/µL (an average of 91.3 ± 3.6 µg/µL).

Figure 5 Standard curves relating the growth of Escherichia coli strain CAG12185 to the known concentration of ornithine added to cultures containing wild-type mouse urine (panel A) or serum (panel B). These standard curves correspond to the standard assays shown in Figure 3, panels E and F. Turbidity in the culture wells at 48 hours of incubation (indicative of bacterial growth and based on the average of 3 measures of absorbance at 600 nm) was plotted against the known concentrations of ornithine present in the microtiter well. A 3-parameter sigmoidal logistic regression was generated from the plotted values to produce an equation relating ornithine concentration to growth. From the equation and the absorbance readings resulting from bacterial growth in the physiological fluid samples gathered from mutant rhg/rhg or rhg/Oat^∆ mice, ornithine concentrations in the respective physiological fluid samples were determined (see text).

Figura 6 Rescue of the auxotrophic, argE mutant Escherichia coli strain BW6165 on M9 minimal media by addition of exogenous ornithine or physiological fluids from mouse mutants with hyperornithinemia. A and B, E coli BW6165 was cultured in microtiter wells that contained M9 minimal glucose media and wild-type urine (panel A) or serum (panel B) that had been spiked with various concentrations of ornithine, as indicated. In both standard assays, rescue of bacterial growth was proportional to the concentration of ornithine supplied. In panels C and D, E coli BW6165 was cultured in microtiter wells that contained M9 minimal glucose media and urine or serum, respectively, from mice with the 5 genotypes indicated. In both experimental assays, rhg/Oat^∆ urine or serum samples rescued more growth than rhg/rhg samples, but although urine from +/+. +/rhg, or +/Oat^∆ mice rescued no bacterial growth in these cultures, serum samples from wild-type mice with these genotypes sometimes stimulated modest increases in turbidity (eg, see +/rhg culture in panel D). Similar results were obtained in microtiter culture experiments that used E coli strain CAG12185 (see Figure 3). E and F, Standard curves relating the growth of E coli strain BW6165 to known concentrations of ornithine added in wild-type mouse urine (panel E) or serum (panel F), based on the average of 3 turbidity readings taken at 48 hours of incubation in the same assays shown in panels A and B, respectively. A 3-parameter sigmoidal logistic regression was generated from the plotted values to produce an equation relating ornithine concentration to growth. With this equation, bacterial growth observed in the experimental assays of mutant (rhg/rhg or rhg/Oat^∆) urine (panel C) or serum (panel D) can be interpreted as ornithine concentrations in the respective physiological fluid samples. From the standard curve shown in panel E, the ornithine concentration in urine from mutant rhg/rhg mice was 3.4 ± 0.2 mmol/L (N = 3) and from rhg/Oat^∆ mice was 4.7 ± 0.4 mmol/L (N = 3), in close agreement with values determined using E coli strain CAG12185 (see text). From the standard curve shown in panel F, the ornithine concentrations in mutant serum samples were found to be 1015 ± 35 µmol/L for rhg/rhg (N = 3) and 1139 ± 69 µmol/L for rhg/Oat^∆ serum (N = 3), again in good agreement with values determined using E coli CAG12185 and with values measured directly by Bisaillon et al¹⁰10 Bisaillon, JJ, Radden, LA II, Szabo, ET. The retarded hair growth (rhg) mutation in mice is an allele of ornithine aminotransferase (Oat). Mol Genet Metab Rep. 2014;1:378–390. (see text).

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Auxotrophy-Based Detection of Hyperornithinemia in Mouse Blood and Urine

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[1] Corresponding Author: Thomas R. King, PhD, Biomolecular Sciences, Central Connecticut State University, 1615 Stanley Street, New Britain, CT 06053, USA. Email: kingt@ccsu.edu