Classical homocystinuria (HCU; CβS deficiency) is characterized by a blockage in homocysteine (Hcy) degradation, resulting in Hcy and methionine accumulation and cysteine deficiency. Studies in healthy and chronically ill individuals have found positive associations between proinflammatory cytokines and plasma total homocysteine (tHcy) [[1], [2], [3]], suggesting a role for immunomodulation in HCU pathogenesis. Therefore, we aimed to investigate 20 inflammatory cytokines in plasma of poorly controlled HCU patients and healthy controls.
The study sample comprised 9 late-diagnosed HCU patients and 10 age and gender-matched healthy controls from South Brazil. tHcy, cysteine, methionine, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were measured in plasma by LC-MS/MS. The cytokines quantification assay was performed through EMD Millipore's MILLIPEX® MAP Human Cytokine kit, accordingly manufacturer's instruction. All samples were measured in duplicates for 20 cytokines (Table 1). Measurements with divergence ≥30% between duplicates were excluded from data analysis (n = 1).
Table 1.
Homocysteine-related metabolites and cytokine levels in HCU patients and controls.
|
Patients (n = 9) |
Controls (n = 10) |
p | ||
|---|---|---|---|---|
| Median (range) | Median (range) | |||
| Homocysteine-related metabolites (μmol/L) | Met | 165 (22–777) | 24 (14–30) | 0.007 |
| Hcy | 130 (17–300) | 6.7 (5.7–12) | <0.001 | |
| Cys | 158 (67–297) | 223 (174–241) | 0.014 | |
| SAM | 756 (99–3264) | 82 (69–107) | <0.001 | |
| SAH | 135 (18–591) | 19 (10−23) | 0.002 | |
| Pro-inflammatory cytokines (pg/mL) | IL-1α | 0.05 (0.01–1.25) | 0.24 (0.01–30.77) | 0.252 |
| IL-1β | 0.79 (0.39–1.83) | 0.57 (0.37–1.05) | 0.743 | |
| IL-6 | 1.03 (0.45–2.05) | 1.12 (0.47–11.56) | 0.653 | |
| IL-8 | 2.07 (0.58–15.60) | 1.58 (1.12–18.14) | 0.624 | |
| IL-17 | 1.96 (1.04–5.48) | 4.09 (1.23–12.94) | 0.102 | |
| TNF-α | 7.92 (3.62–14.81) | 9.17 (3.92–13.67) | 0.287 | |
| TNF-β | 0.01 (0.00–1.94) | 0.02 (0.00–119) | 0.617 | |
| MCP-1 | 254 (185–1014) | 267 (216–357) | 0.935 | |
| IP-10 | 277 (170–1855) | 489 (264–1764) | 0.153 | |
| GRO | 829 (197–2473) | 704 (303–1014) | 0.595 | |
| MDC | 554 (299–1288) | 527 (295–759) | 0.744 | |
| MIP-1α | 1.00 (0.54–3.62) | 2.24 (0.66–34.89) | 0.077 | |
| MIP-Iβ | 18.61 (1.01–37.62) | 19.57 (1.84–68.80) | 0.327 | |
| VEGF | 156.71 (0.01–376) | 255.33 (1.32–704) | 0.102 | |
| GM-CSF | 3.62 (1.06–15.20) | 3.01 (1.77–13.67) | 0.744 | |
| IFN-γ | 3.62 (1.67–9.72) | 10.92 (4.94–82.04) | 0.007 | |
| Anti-inflammatory cytokines (pg/mL) | IL-4 | 0.48 (0.04–2.83) | 0.68 (0.16–26.46) | 0.327 |
| IL-10 | 0.75 (0.27–1.39) | 0.79 (0.41–3.29) | 0.935 | |
| IL-13 | 0.11 (0.02–9.17) | 0.15 (0.02–78) | 0.368 | |
| G-CSF | 13.67 (7.69–46.01) | 16.83 (7.69–135.66) | 0.653 |
Met: methionine; Hcy: homocysteine; Cys: cysteine; SAM: S-Adenosylmethionine; SAH: S-Adenosylhomocysteine.
Hcy-lowering treatment (pyridoxine 7/9, folic acid 8/9, betaine 7/9, methionine-restricted diet 3/9) was prescribed; but only 3/9 patients had tHcy <100 μmol/L (target level). Most patients (7/9) were pyridoxine nonresponsive. Because of the high concentrations of tHcy with a wide range we consider this an ideal group to explore the potential relation between cytokines and tHcy. Cytokines plasma levels were similar in patients and controls, with the exception of IFN-γ, which was three-fold reduced (p = .007) in patients (Table 1). In line an inverse association of Hcy and SAM with IFN-γ was found (r − 0.487 and r − 0.537; p < .05).
To our knowledge, only one study had previously evaluated cytokines in HCU patients. Keating et al. measured 16 cytokines in plasma of HCU patients, and found that patients with tHcy >150 μM, (n = 5) had increased levels of several pro-inflammatory cytokines (IL-1a, IL-6, TNF-α, IL-17 and IL-12), while well controlled patients (Hcy <86.1 μM, n = 5) had not [4]. The authors provided no information about which patients received treatment. IFN-γ was not evaluated in this study. In previous studies, reduced IFN-γ levels have shown anti-inflammatory properties [5,6].
In summary, our study provides no evidence of increased inflammatory cytokines in HCU patients on treatment, despite poor metabolic control. Hcy may even show anti-inflammatory properties like glutathione [7], what could explain the finding of lowered IFN-γ. The potential impact of Hcy-lowering treatment on cytokines requires further study.
Acknowledgments
Financial support for this study was provided by CNPq, FAPERGS, CAPES and DAAD.
References
- 1.Gori A.M., Corsi A.M., Fedi S., Gazzini A., Sofi F., Bartali B., Bandinelli S., Gensini G.F., Abbate R., Ferrucci L. A proinflammatory state is associated with hyperhomocysteinemia in the elderly. Am. J. Clin. Nutr. 2005;82:335–341. doi: 10.1093/ajcn.82.2.335. [DOI] [PubMed] [Google Scholar]
- 2.Shai I., Stampfer M.J., Ma J., Manson J.E., Hankinson S.E., Cannuscio C., Selhub J., Curhan G., Rimm E.B. Homocysteine as a risk factor for coronary heart diseases and its association with inflammatory biomarkers, lipids and dietary factors. Atherosclerosis. 2004;177:375–381. doi: 10.1016/j.atherosclerosis.2004.07.020. [DOI] [PubMed] [Google Scholar]
- 3.Michaud M., Balardy L., Moulis G., Gaudin C., Peyrot C., Vellas B., Cesari M., Nourhashemi F. Proinflammatory cytokines, aging, and age-related diseases. J. Am. Med. Dir. Assoc. 2013;14:877–882. doi: 10.1016/j.jamda.2013.05.009. [DOI] [PubMed] [Google Scholar]
- 4.Keating A.K., Freehauf C., Jiang H., Brodsky G.L., Stabler S.P., Allen R.H., Graham D.K., Thomas J.A., Van Hove J.L., Maclean K.N. Constitutive induction of pro-inflammatory and chemotactic cytokines in cystathionine beta-synthase deficient homocystinuria. Mol. Genet. Metab. 2011;103:330–337. doi: 10.1016/j.ymgme.2011.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Flaishon L., Topilski I., Shoseyov D., Hershkoviz R., Fireman E., Levo Y., Marmor S., Shachar I. Cutting edge: anti-inflammatory properties of low levels of IFN-gamma. J. Immunol. 2002;168:3707–3711. doi: 10.4049/jimmunol.168.8.3707. [DOI] [PubMed] [Google Scholar]
- 6.Mühl H., Pfeilschifter J. Anti-inflammatory properties of pro-inflammatory interferon-gamma. Int. Immunopharmacol. 2003;3:1247–1255. doi: 10.1016/S1567-5769(03)00131-0. [DOI] [PubMed] [Google Scholar]
- 7.Pirchl M., Ullrich C., Sperner-Unterweger B., Humpel C. Homocysteine has anti-inflammatory properties in a hypercholesterolemic rat model in vivo. Mol. Cell. Neurosci. 2012;49:456–463. doi: 10.1016/j.mcn.2012.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]