Effect of standard phlebotomy on myocardial and hepatic iron levels in newly diagnosed cardiac asymptomatic hereditary hemochromatosis subjects with C282Y homozygosity

Standard phlebotomy effectively reduces systemic and tissue iron overload (IO) in hemochromatosis patients [1–3]. However, the long-term therapeutic effect ofphlebotomy on tissue IOinnewlydiagnosed cardiac asymptomatic hereditary hemochromatosis (HH) subjects during the course of standard therapy in a prospective cohort is not described. Up to now, only one cohort study examined changes in myocardial iron levels after phlebotomy therapy in newly diagnosed HH subjects as far as we know [4]. The study showed myocardial iron levels did not change significantly after 3 months of the standard therapy in the newly diagnosed HH subjects; however, it lacked longer termfollow-up,waslimitedtomyocardialiron,andhadnocontrolsub-jects. Therefore, we have examined the effect of standard phlebotomy therapy on myocardial and hepatic iron content measured with T2* (T2 star magnetic vector decay constant) derived from noninvasive magnetic resonance imaging (MRI) in our cohort of cardiac asymptomatic HH subjects ( n = 22) with C282Y homozygosity as well as in age-gender matched normal volunteers (NV, n = 21) who lacked the HFE mutation to cause HH over a 5-year follow-up in the National, Heart, Lung, and Blood Institute (NHLBI)-sponsored “Heart Study of Hemochromatosis” (ClinicalTrials.gov number, NCT00068159) [5–7]. The study


Effect of standard phlebotomy on myocardial and hepatic iron levels in newly diagnosed cardiac asymptomatic hereditary hemochromatosis subjects with C282Y homozygosity
Standard phlebotomy effectively reduces systemic and tissue iron overload (IO) in hemochromatosis patients [1][2][3]. However, the longterm therapeutic effect of phlebotomy on tissue IO in newly diagnosed cardiac asymptomatic hereditary hemochromatosis (HH) subjects during the course of standard therapy in a prospective cohort is not described. Up to now, only one cohort study examined changes in myocardial iron levels after phlebotomy therapy in newly diagnosed HH subjects as far as we know [4]. The study showed myocardial iron levels did not change significantly after 3 months of the standard therapy in the newly diagnosed HH subjects; however, it lacked longer term follow-up, was limited to myocardial iron, and had no control subjects. Therefore, we have examined the effect of standard phlebotomy therapy on myocardial and hepatic iron content measured with T2* (T2 star magnetic vector decay constant) derived from noninvasive magnetic resonance imaging (MRI) in our cohort of cardiac asymptomatic HH subjects (n = 22) with C282Y homozygosity as well as in age-gender matched normal volunteers (NV, n = 21) who lacked the HFE mutation to cause HH over a 5-year follow-up in the National, Heart, Lung, and Blood Institute (NHLBI)-sponsored "Heart Study of Hemochromatosis" (ClinicalTrials.gov number, NCT00068159) [5][6][7].
The study was approved by the Institutional Review Board of the NHLBI of the National Institutes of Health (NIH) and was performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards [8]. Consent for the study was obtained from the participants. The demographic data of these groups were published in our previous articles [5,7]. The average age of the newly diagnosed HH group was 48 ± 11 years (data are mean ± SD, 27% female) and that of the NV group was 48 ± 8 years at baseline (33% female). They showed no significant left ventricular systolic dysfunction as compared to NV subjects [5,7]. The research nurses for this study communicated with the HH subjects throughout the study period to ensure the mainte- Center. T2* of myocardium and liver was measured from the images obtained with a 1.5 T MRI scanner using a gradient echo sequence [9,10]. Smaller T2* measurements represent higher tissue iron level.
MRIs were performed at baseline, 6-month, and 5-year follow-ups in Among newly diagnosed HH subjects whose tissue T2*were evaluable, the measures of systemic IO as well as alanine aminotransferase and aspartate aminotransferase were significantly improved over time by standard phlebotomy therapy ( Table 1).
This study is the first to systematically show that both myocardial and hepatic IO were prevented or improved in the newly diagnosed HH cohort subjects with standard phlebotomy. The myocardial iron levels of HH subjects were comparable to age-gender matched NV subjects throughout the study period of 5 years. The hepatic iron level was elevated at baseline in the HH population; however, it normalized to the level of NV subjects after 5 years of conventional phlebotomy therapy. We have reported persistently elevated oxidative stress levels [11] in this population despite their normal myocardial tissue iron levels while receiving standard phlebotomy therapy [6,12] and speculated that the elevated oxidative level may potentially be responsible for long-term tissue damage related to HH [11,13,14]. Thus, there still remains a concern for long-term tissue damage in the HH population.
This study has several limitations including the small cohort size, the use of only MRI to measure tissue iron levels, and being limited to HH with C283Y homozygosity. In addition, the standard of phlebotomy therapy might vary locally; however, our blood testing results assure that HH subjects were consistent with the accepted therapeutic effect of maintenance standard phlebotomy therapy [3,15].
In conclusion, our prospective study demonstrated that conventional phlebotomy therapy over 5 years was effective in pre-

CONFLICT OF INTEREST STATEMENT
The authors have no conflict of interest to disclose.

FUNDING INFORMATION
National Heart, Lung, and Blood Institute of the National Institutes of Health

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are governed by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health. This study protocol does not have the approved data sharing plan by the NHLBI. However, a data sharing request may be submitted to the NHLBI for consideration on a case-by-case basis.

ETHICS STATEMENT
The study was approved by the Institutional Review Board of the NHLBI of the National Institutes of Health (NIH) and was performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.
Consent for the study was obtained from the participants.