Haematologica
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Published online 26 January 2008
Haematologica, Vol 93, Issue 2, 279-282 doi:10.3324/haematol.11851
Copyright © 2008 by Ferrata Storti Foundation
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Erythrocytosis

Elevated homocysteine, glutathione and cysteinylglycine concentrations in patients homozygous for the Chuvash polycythemia VHL mutation

Adelina I. Sergueeva1, Galina Y. Miasnikova2, Daniel J. Okhotin4, Alla A. Levina3, Zufan Debebe7, Tatiana Ammosova7, Xiaomei Niu7, Elena A. Romanova3, Sergei Nekhai7, Patricia M. DiBello5, Donald W. Jacobsen5, Josef T. Prchal6, Victor R. Gordeuk7,

1 Cheboksary Children’s Hospital, Cheboksary, Russia;
2 Chuvash Republic Clinical Hospital No. 1, Chuvashia, Russia;2
3 Hematology Center, Moscow, Russia;
4 Russian Research Services, San Diego, CA, USA;
5 Cleveland Clinic, Cleveland, OH, USA;
6 University of Utah, Salt Lake City, UT, USA and
7 Howard University, Washington, DC, USA

Correspondence: Victor R. Gordeuk, M.D., Howard University, 2041 Georgia Ave NW, Washington, DC 20060 USA. E-mail: vgordeuk{at}Howard.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Design and Methods
 Results and Discussion
 Discussion and Results
 References
 
In Chuvash polycythemia, homozygous von Hippel-Lindau (VHL) 598C>T leads to increased hypoxia inducible factor-1{alpha} and 2{alpha}, thromboses and lower systemic blood pressures. Circulating homocysteine, glutathione, {gamma}-glutamyltransferase and cysteinylglycine concentrations were higher in 34 VHL598C>T homozygotes than in 37 normal controls and cysteine was lower. Multivariate analysis showed elevated homocysteine independently associated with higher mean systemic blood pressures and elevated glutathione was associated with lower pressures to a similar degree. Among VHL598C>T homozygotes, homocysteine was elevated with low and normal folate concentrations, consistent with a possible defect in the remethylation pathway. The elevated glutathione and {gamma}-glutamyltranserase levels correlated positively with cysteinylglycine, consistent with possible upregulation of a glutathione synthetic enzyme and {gamma}-glutamyltransferase. Cysteinylglycine correlated inversely with cysteine, consistent with possible reduced cysteinyldipeptidase activity. We conclude that up-regulated hypoxia-sensing may influence multiple steps in thiol metabolism. The effects of the resultant elevated levels of homocysteine and glutathione on systemic blood pressure may largely balance each other out.

Key words: VHL, polycythemia, homocysteine, folate, glutathione.


   Introduction
TOP
 ABSTRACT
 Introduction
Design and Methods
 Results and Discussion
 Discussion and Results
 References
 
Chuvash polycythemia, the first recognized congenital defect of augmented hypoxia-sensing, is characterized by homozygous von Hippel-Lindau germ-line mutation (VHL598C>T)1, upregulation of hypoxia inducible factor (HIF)-1{alpha}, HIF-2{alpha} and several target genes under normoxia,1,2 arterial and venous thrombosis,2 lower systemic blood pressures,2 elevated pulmonary artery pressures3 and other abnormalities in respiratory and pulmonary vascular regulation.4 Many other genes are regulated by HIF-15 and might have altered expression. Mutations of prolylhydroxylase-2 also lead to upregulation of HIF in normoxia.6 Elevated plasma homocysteine levels are associated with an increase in blood pressure and with thrombosis.7,8 Lower cellular glutathione levels have been associated with hypertension.9 Homocysteine levels depend on remethylation of homocysteine to methionine (with methylene tetrahydrofolate reductase (MTHFR) and methionine synthase) and/or transsulfuration to cysteine (with cystathionine beta-synthase and cystathionine gammalyase).10

Cysteine is the rate-limiting amino acid for synthesis of the antioxidative tripeptide, glutathione, which involves gamma-glutamylcysteine synthetase and glutathione synthetase. Catabolism of glutathione leads to formation of cysteinylglycine through the action of gamma-glutamyl transpeptidase (GGT). Cysteinylglycine can be catabolized through the action of a dipeptidase to cysteine.


   Design and Methods
TOP
 ABSTRACT
 Introduction
 Design and Methods
Results and Discussion
 Discussion and Results
 References
 
The research was approved by Howard University’s Institutional Review Board. All study subjects provided written informed consent. Serum folic acid, vitamin B12 and GGT concentrations were determined by Quest Diagnostics (Baltimore, MD, USA). Total plasma cysteine, glutathione and cysteinylglycine concentrations were determined by high performance liquid chromatography (HPLC) with fluorescence detection.11 Reference ranges are: homocysteine: 3.3–13.9 uM; cysteine: 136–308 uM; glutathione: 4.1–9.9 uM; cysteinylglycine: 20.5–41.0 uM.11,12 Polymerase chain reactionPCR for the VHL598C>T mutation was performed as reported.3 MTHFR677T>C was detected by allele-specific polymerase chain reaction. Amplification of MTHFR exon 4 was carried out in 50 µL PCR master mix containing 1.0 U of Taq DNA Polymerase (Promega, USA), 150 ng DNA and 1.5 pmol of each of the following primers: 5'-CTCTCTCTCTGAAGGAGAAGGTGTCTGCGGGA-3' and 5'-AGGACGGTGCGGTGAGAGTG-3'. Each reaction was controlled by a pair of primers that amplified a 429 bp fragment of human growth hormone gene. Conditions were denaturation for three min, at 95°C, followed by 35 cycles at 95°C for 15 sec, 61°C for 15 sec, 72°C for 15 sec, and a final extension step of ten min at 72°C. PCR products were separated by electrophoresis on 2% agarose gel.

Results were compared according to VHL or MTHFR genotype using ANOVA (continuous variables) or Pearson’s {chi}2 test (categorical variables). Linear regression was used to evaluate relationships among homocysteine, cysteine, glutathione, cysteinylglycine, GGT and other important variables. Systat® software (San José, CA, USA) was used. Outlying values or values with large leverage were excluded from the ANOVA and linear regression models.


   Results and Discussion
TOP
 ABSTRACT
 Introduction
 Design and Methods
 Results and Discussion
Discussion and Results
 References
 
General characteristics and thiol concentrations of study participants
Thirty-five patients with the diagnosis of Chuvash polycythemia (n=16, <20 years of age) and 40 Chuvash control subjects without polycythemia (n=17, <20 years) were studied. Thirty-four patients with a diagnosis of Chuvash polycythemia were VHL598C>T homozygotes and one had normal VHL alleles. Thirty-six controls had normal VHL alleles and four were VHL598C>T heterozygotes. Fourteen out of 34 VHL 598C>T homozygotes had a history of phlebotomy therapy and six had normal or low hemoglobin concentrations at the time of study. One of the VHL 598C>T homozygotes had a history of peripheral thrombosis and one participant with normal alleles had a history of myocardial infarction. None of the study subjects had a history of cerebral vascular accident. Serum vitamin B12 levels did not differ significantly according to VHL genotype but folate levels were significantly lower in VHL598C>T homozygotes. Plasma homocysteine, glutathione and cysteinylglycine concentrations were higher in VHL598C>T homozygotes than in subjects with normal VHL genotype and plasma cysteine concentrations were lower both in unadjusted analysis and after adjustment for significant covariates. Adjusted serum GGT concentrations were significantly higher in VHL598C>T homozygotes (Table 1, online supplement).

Relationship of plasma thiol concentrations to systemic blood pressure
In a linear regression analysis, mean systemic blood pressure correlated independently and positively with plasma homocysteine concentration (p=0.039) as well as with age (p<0.001) and hemoglobin concentration (p=0.013). At the same time, mean pressure correlated independently and negatively with VHL598C>T (p=0.008) and plasma glutathione concentration (p=0.015) (Table 2, online supplement). After adjustment for the other covariates, a 6.6 uM increase in plasma homocysteine concentration (the average increase between subjects with normal alleles and VHL598C>T homozygotes in the present study shown in Table 1, online supplement) was associated with an estimated 3 mm Hg increase in mean blood pressure (95% confidence interval of 0.1 to 5 mm Hg increase in mean blood pressure). After adjustment for the other covariates, a 6.8 µM increase in plasma glutathione concentration (the average increase between participants with normal alleles and VHL598C>T homozygotes in the present study) was associated with an estimated 3 mm Hg (0.5 to 5 mm Hg) decrease in mean blood pressure.

Homocysteine
Univariate analysis of subjects with normal VHL genotype, showed plasma homocysteine concentrations to have the expected inverse correlations with both serum vitamin B12 levels (n=33, R=–0.627, p<0.001) and serum folate levels. Among VHL 598C>T homozygotes, plasma homocysteine concentrations had an inverse correlation with serum vitamin B12 (n=31, R=–0.389, p=0.031) but serum folate did not. Indeed, homocysteine tended to be elevated regardless of the folate concentration. There was no significant difference between plasma homocysteine concentrations according to MTHFR677C>T polymorphism status (p=0.4).

Cysteine
Plasma cysteine concentration had a positive relationship with homocysteine concentration in both subjects with normal VHL genotype (n=36, R=0.66, p<0.001) and VHL598C>T homozygotes (n=32, R=0.29, p=0.1). After adjustment for cysteinylglycine concentration and age category, the positive relationship between cysteine and homocysteine was maintained in both VHL 598C>T homozygotes (p=0.0002) and subjects with normal VHL alleles (p=0.0004). In contrast, cysteine had a positive relationship with cysteinylglycine in VHL wildtype subjects (n=37, R=0.33, p=0.047) and a negative relationship in VHL598C>T homozygotes (N=34, R=–0.27, p=0.1). After adjustment for homocysteine concentration and age category, there was a significant inverse relationship between cysteinylglyine and cysteine in VHL598C>T homozygotes (p=0.0005) but not in subjects with normal alleles (p=0.9).

Glutathione
There was no significant association between plasma glutathione concentration and cysteine concentration in either wildtype subjects or VHL598C>T homozygotes. Glutathione correlated significantly with cysteinylglycine in VHL598C>T homozygotes (n=34, R=0.60, p=0.0002) but not in subjects with normal VHL alleles (n=36, R=0.04, p=0.8).

Cysteinylglycine
Plasma cysteinylglycine concentration did not correlate significantly with either glutathione or GGT in subjects with wildtype VHL alleles. However, cysteinylglycine correlated positively with glutathione after adjustment for GGT level and age category (p=0.001) and with GGT after adjustment for glutathione and age category (p=0.023) in VHL598C>T homozygotes. Cysteinylglycine levels correlated significantly with homocysteine levels in both VHL598C>T homozygotes (n=34, R=0.54, p=0.001) and individuals with normal VHL genotype (n=36, R=0.54, p<0.001).


   Discussion and Results
TOP
 ABSTRACT
 Introduction
 Design and Methods
 Results and Discussion
 Discussion and Results
References
 
Circulating concentrations of homocysteine, glutathione, {gamma}-glutamyltransferase and cysteinylglycine were elevated and cysteine concentrations were reduced in homozygotes for the VHL598C>T Chuvash polycythemia mutation. Our observations suggest that multiple steps in thiol metabolism may be influenced by HIF-mediated hypoxia sensing. HIF-1 leads to up regulation of certain classes of genes and down regulation of others.5 We are not aware of published evidence that the promoter regions of the genes involved in thiol metabolism have HIF recognition elements.

Elevated homocysteine concentrations are associated with an increased risk of arteriosclersosis, thrombosis, hypertension and renal dysfunction.7,8 Thrombosis is a common complication of Chuvash polycythemia. However, we were unable to test for an association between homocysteine levels and thrombosis in the present study because of the low prevalence of thrombotic events. Another characteristic of Chuvash polycythemia is a tendency to low rather than elevated blood pressures.2 Increased homocysteine7 and depletion of glutathione9 have been associated with an increase in blood pressure. In the present study, higher homocysteine concentrations were associated with higher systemic blood pressures while higher glutathione concentrations were associated with lower pressures to a similar degree (Table 2, online supplement). Therefore, the effects of the elevated levels of homocysteine and glutathione on systemic blood pressure may have largely balanced each other out.

In the present study serum folate levels were significantly lower in VHL598C>T homozygotes (Table 1) but their elevated plasma homocysteine concentrations in VHL598C>T homozygotes were maintained after adjusting for serum folate and vitamin B12 concentrations. An expected inverse correlation between serum folate concentration and plasma homocysteine concentration was found in VHL normal subjects but not VHL598C>T homozygotes, in whom homocysteine levels were elevated with the entire spectrum of folate concentrations. Multivariate analyses showed significant positive correlations between plasma homocysteine and cysteine concentrations in both subjects with VHL wildtype alleles and VHL598C>T homozygotes. Therefore, the elevated homocysteine concentrations might be due to a reduction in the activity of a remethylation pathway enzyme rather than a transsulfuration enzyme.

Elevated homocysteine is usually associated wih increased plasma homocysteine concentration and decreased cysteine concentration due to defects in either the remethylation or transsulfuration pathways.13–15 Elevated plasma homocysteine concentrations have been reported in polycythemia vera and other myeloproliferative disorders,16 but a number of VHL598C>T homozygotes in the present study had normal hematocrits maintained by therapeutic phlebotomy, and homocysteine levels remained elevated after adjustment for hemoglobin concentration (p=0.011).

Plasma glutathione is mainly found in the liver.17–19 The increased glutathione and cysteinylglycine concentrations (Table 1) and the positive correlation of cysteinylglycine with both glutathione and GGT in VHL598C>T homozygotes in the present study suggest that a glutathione synthetic enzyme and GGT may be up-regulated. Notably, GGT levels are elevated in patients with chronic obstructive lung disease,20 GGT is necessary for the normal respiratory response to hypoxia,21 and VHL598C>T homozygotes have both a reduced PCO2 set point for respiratory control and an abnormally high acute hypoxic ventilatory sensitivity.4

The significant negative correlation of plasma cysteine with cysteinylglycine in VHL598C>T homozygotes but not subjects with normal alleles in the present study might be explainable by a reduction in the activity of a cysteinylglycine dipeptidase. The strong positive relationships between plasma homocysteine and plasma cysteinylglycine concentrations suggest a possible common or parallel regulatory mechanism.


   Footnotes
 
The online version of this article contains a supplemental appendix.

Authorship and Disclosures

AIS developed the hypothesis for this study and participated in designing the study, conducting the study, interpreting the results and drafting the manuscript; GYM participated in developing the hypothesis of the study, designing the study, conducting the study, interpreting the results and drafting the manuscript; DJO participated in designing the study, conducting the study and drafting the manuscript; AAL participated in developing the hypothesis, designing the study, conducting the study, interpreting the results and drafting the manuscript; ZD, TA, XN, SN: participated in conducting the study, interpreting the results and drafting the manuscript; EAR participated in developing the hypothesis, conducting the study, interpreting the results and drafting the manuscript; PMDB participated in developing the hypothesis, conducting the study, interpreting the results and drafting the manuscript; DWJ participated in developing the hypothesis, conducting the study, interpreting the results and drafting the manuscript; JTP participated in designing the study, developing the hypothesis, interpreting the results and drafting the manuscript; VRG designed the study and drafted teh manuscript: he participated in developing the hypothesis, conducting the study and interpreting the results.

Funding: this work was supported in part by NIH research grants n. UH1-HL03679 and n. 5 R25-HL02679 from the National Heart, Lung and Blood Institute and the Office of Research on Minority Health, Howard University General Clinical Research Center Grant n. MO1-RR10284, and NIH grant n. 5 RO1 HL079912-02 (VRG), R01HL66333-01 (JTP&VRG) and R01HL5007-13 (JTP).

Received for publication June 14, 2007. Accepted for publication September 12, 2007.


   References
TOP
 ABSTRACT
 Introduction
 Design and Methods
 Results and Discussion
 Discussion and Results
 References
 

  1. Ang SO, Chen H, Hirota K, Gordeuk VR, Jelinek J, Guan Y, et al. Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia. Nat Genet 2002;32:614-21.[CrossRef][ISI][Medline]
  2. Gordeuk VR, Sergueeva AI, Miasnikova GY, Okhotin D, Voloshin Y, Choyke PL, et al. Congenital disorder of oxygen-sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood 2004;103:3924-32.[Abstract/Free Full Text]
  3. Bushuev VI, Miasnikova GY, Sergueeva AI, Polyakova LA, Okhotin D, Gaskin PR, et al. Endothelin-1, vascular endothelial growth factor and systolic pulmonary artery pressure in patients with Chuvash polycythemia. Haematologica 2006;91:744-9.[Abstract/Free Full Text]
  4. Smith TG, Brooks JT, Balanos GM, Lappin TR, Layton DM, Leedham DL, et al. Mutation of von Hippel-Lindau tumour suppressor and human cardiopulmonary physiology. PLoS Med 2006;3:e290.[CrossRef][Medline]
  5. Manalo DJ, Rowan A, Lavoie T, Natarajan L, Kelly BD, Ye SQ, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 2005;105:659-69.[Abstract/Free Full Text]
  6. Percy MJ, Furlow PW, Beer PA, Lappin TR, McMullin MF, Lee FS. A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove. Blood 2007;110:2193-6.[Abstract/Free Full Text]
  7. Refsum H, Nurk E, Smith AD, Ueland PM, Gjesdal CG, Bjelland I, et al. The Hordaland Homocysteine Study: a community-based study of homocysteine, its determinants, and associations with disease. J Nutr 2006;136 Suppl_6: 1731S-1740S.[Abstract/Free Full Text]
  8. Martinelli I, Battaglioli T, Pedotti P, Cattaneo M, Mannucci PM. Hyper-homocysteinemia in cerebral vein thrombosis. Blood 2003;102:1363-6.[Abstract/Free Full Text]
  9. Levonen AL, Laakso J, Vaskonen T, Mervaala E, Karppanen H, Lapatto R. Down-regulation of renal glutathione synthesis by systemic nitric oxide synthesis inhibition in spontaneously hypertensive rats. Biochem Pharmacol 2000;59:441-3.[CrossRef][ISI][Medline]
  10. Finkelstein JD. The metabolism of homocysteine: Pathways and regulation. Eur J Pediatr 1998;157 Suppl: S40-4.[CrossRef][ISI][Medline]
  11. Jacobsen DW, Gatautis VJ, Green R, Robinson K, Savon SR, Secic M, et al. Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlations with cobalamin and folate levels in normal subjects. Clin Chem 1994;40:873-81.[Abstract/Free Full Text]
  12. Mansoor MA, Svardal AM, Schneede J, Ueland PM. Dynamic relation between reduced, oxidized, and protein-bound homocysteine and other thiol components in plasma during methionine loading in healthy men. Clin Chem 1992;38:1316-21.[Abstract/Free Full Text]
  13. Kraus JP, Janosik M, Kozich V, Mandell R, Shih V, Sperandeo MP, et al. Cystathionine b-synthase mutations in homocystinuria. Hum Mutat 1999;13:362-75.[CrossRef][ISI][Medline]
  14. Blaise S, Alberto JM, Nedelec E, Ayav A, Pourie G, Bronowicki JP, et al. Mild neonatal hypoxia exacerbates the effects of vitamin-deficient diet on homocysteine metabolism in rats. Pediatr Res 2005;57:777-82.[CrossRef][ISI][Medline]
  15. Ranganath LR, Baines M, Roberts NB. Homocysteine and thiol metabolites in vitamin B12 deficiency. Clin Sci (Lond) 2001;100:111-6.[Medline]
  16. Amitrano L, Guardascione MA, Ames PR, Margaglione M, Antinolfi I, Iannaccone L, et al. Thrombophilic genotypes, natural anticoagulants, and plasma homocysteine in myeloproliferative disorders: relationship with splanchnic vein thrombosis and arterial disease. Am J Hematol 2003;72:75-81.[CrossRef][ISI][Medline]
  17. Lu SC. Regulation of hepatic glutathione synthesis: current concepts and controversies. FASEB J 1999;13:1169-83.[Abstract/Free Full Text]
  18. Griffith OW, Meister A. Translocation of intracellular glutathione to membrane-bound g-glutamyl-transpeptidase as a discrete step in the g-glutamyl cycle: glutathionuria after inhibition of transpeptidase. Proc Natl Acad Sci USA 1979;76:268-72.[Abstract/Free Full Text]
  19. Adams JD Jr, Lauterburg BH, Mitchell JR. Plasma glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress. J Pharmacol Exp Ther 1983;227:749-54.[Abstract/Free Full Text]
  20. Barcelo A, Barbe F, de la Pena M, Vila M, Perez G, Pierola J, et al. Anti-oxidant status in patients with sleep apnoea and impact of continuous positive airway pressure treatment. Eur Respir J 2006;27:756-60.[Abstract/Free Full Text]
  21. Lipton AJ, Johnson MA, Macdonald T, Lieberman MW, Gozal D, Gaston B. S-nitrosothiols signal the ventilatory response to hypoxia. Nature 2001;413:171-4.[CrossRef][Medline]




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