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FRANCESCO S. FACCHINIa,b,c AND KAMI L. SAYLORb aDepartment of Medicine, Division of Nephrology, San Francisco General Hospital and University of California, San Francisco, California, USA bDepartment of Medicine, Kaiser Foundation Hospitals, and Permanente Medical Group, Incorporated, Oakland, California, USA cZentrum für Schlafmedizin und Stoffwechselstörungen, Dortmund, Germany

ABSTRACT: Controversy surrounds the role of iron (Fe) in atherosclerosis (ASCVD), mainly due to the inaccuracy of assessing body Fe stores with and saturation. Quantitative phlebotomy was used to test whether or not (a) Fe stores are increased in individuals at high risk for ASCVD and (b) Fe depletion to near-deficiency (NID) levels is associated with reduction of risk factors for ASCVD. Thirty-one carbohydrate-intolerant sub- jects completed the study. Fe stores were within normal limits (1.5 ± 0.1 g). At NID, a significant increase of HDL- (p < 0.001) and reductions of blood pressure (p < 0.001), total and LDL-cholesterol (p < 0.001), triglyceride (p < 0.001), fibrinogen (p < 0.001) and glucose and insulin responses to oral loading (p < 0.001) were noted, while homocysteine plasma concentra- tion remained unchanged. These effects were largely reversed by a 6-month period of Fe repletion with reinstitution of Fe sufficiency. Thus, although individuals at high risk for ASCVD are not Fe-overloaded, they seem to benefit, metabolically and hemodynamically, from lowering of body Fe to levels commonly seen in premenopausal females.

KEYWORDS: iron; atherosclerosis; hypertension; glucose intolerance; ; ;

INTRODUCTION

In 1981, Sullivan proposed that the gender difference in the incidence of athero- sclerotic cardiovascular disease (ASCVD) might be related to Fe accumulation in men as opposed to premenopausal females.1 Since then, many epidemiological studies tested such a hypothesis with conflicting results.2,3 Methodological bias is likely at the origin of such controversy as dietary Fe intake, iron saturation, and serum ferritin concentration (SFC) are all indirect and unreliable measurements of

Address for correspondence: Dr. Francesco S. Facchini, Box 1341, UCSF, San Francisco, CA 94080-1341. Fax: 510-420-1988. [email protected]

Ann. N.Y. Acad. Sci. 967: 342–351 (2002). © 2002 New York Academy of Sciences.

342 FACCHINI & SAYLOR: IRON DEPLETION 343

Fe stores. Dietary Fe content bears little relation with absorption since Fe bio- availability is either markedly reduced or enhanced by so many factors contained in food.4 (Fe saturation) has a day-to-day variability of about 30%5 and SFC becomes insensitive in the range of 70–250 µg/L, where only 21% of its variance relates to the size of body Fe stores.6 Therefore, relying upon such methodologies cannot resolve whether or not Fe stores are increased in individuals at risk for ASCVD. The most compelling evidence against the hypothesis that body Fe progressively increases with aging is the fact that intestinal absorption of Fe is tightly regulated and, as body Fe increases, absorption rapidly decreases.6,7 Such negative-feedback inhibition seems very efficient, preventing further accumulation of Fe at SFC of ~60 µg/L (corresponding to a total body Fe pool of ~0.5–1.5 g).8 Accordingly, autopsy studies in normal subjects confirmed that, after the fourth decade of life, there is no further increase in liver Fe beyond these levels.8,9 These findings thus argue against the possibility that Fe accumulation occurs in aging individuals and raise the possibility that the protection seen in fertile women from ASCVD may be related to a state of menstruation-induced Fe deficiency or near-iron deficiency10 (NID) rather than overload. Support for this hypothesis comes from studies showing failure of estrogen replacement therapy in protecting postmeno- pausal women from ASCVD,11 as well as from other studies where risk of ASCVD before the age of natural menopause was enhanced by isolated hysterectomy rather than oophorectomy.12 As carbohydrate (CHO) intolerance, dyslipidemia, and hypertension tend to cluster in the same individuals,13 synergistically increasing risk of ASCVD,14–16 a key question would thus be whether or not Fe depletion, to a degree similar to that seen in fertile women, might provide protection against ASCVD by ameliorating such cluster of risk factors. Two small-scale trials partially attempted to address this question in diabetic individuals by lowering body Fe with deferoxamine.17,18 SFC was lowered from a “high” to a “high-normal” level, Fe loss was not quantitated, and opposite results were found, leaving the foregoing question unsettled. The current work originates from the following hypothesis: if there is a beneficial effect secondary to low Fe status, it should best be noted at NID, e.g., that state where Fe depletion is maximal and yet compatible with lack of anemia. Further- more, it would be important to show an eventual beneficial effect in individuals who are at high risk for ASCVD rather than in an unselected sample of patients. Upon these premises, a study was initiated to address the following questions: (1) Are body Fe stores truly increased in patients at high risk for ASCVD? (2) Is SFC a valid surrogate measure of body Fe stores in such patients? (3) Is lowering of Fe stores to NID associated with better ASCVD risk profile?

METHODS

The study was approved by the local ethical committees, experimental procedures were in accordance with institutional guidelines, and informed consent obtained. Patients with either recent type-2 diabetes or impaired glucose tolerance, none of whom were insulin-treated, with no prior history of cardiovascular, retinal, or renal complications were invited to participate. None of the patients had any acute, chronic 344 ANNALS NEW YORK ACADEMY OF SCIENCES inflammatory or neoplastic disease, and fasting glycemia was <7.8 mM on three repeated occasions. Genetic hemochromatosis (GH) was excluded by a saturation < 50% and absent C282Y/H63D.19 After overnight fasting, a 3-h OGTT with hourly measurement of plasma glucose and insulin20 was performed. Fasting total cholesterol, , HDL-cholesterol, HbA1c, , fibrinogen, Fe, transferrin, SFC, and homocysteine were all measured by routine automated techniques. LDL-cholesterol was estimated with Friedewald’s formula.21 Twenty- four-hour, ambulatory blood pressure was measured hourly with a portable monitor- ing device (model TM 2421, A&D Engineering, Milpitas, CA). For analysis, all readings from each monitoring period were averaged for heart rate and systolic and diastolic blood pressure. Quantitative phlebotomy was used to measure body Fe stores.22 Half-liter phlebotomies were performed monthly or bimonthly after topical anesthesia with 0.5–1.0 mL of 1% lidocaine. Fe indexes and a CBC were measured before every phlebotomy to exclude anemia. Hematocrit (Hct) was not allowed to decrease more than 5% (from baseline values) by delaying the next phlebotomy as necessary. Phlebotomies were then discontinued at NID defined as serum ferritin ≤ 30 µg/L, iron saturation ≤ 15%, and mean corpuscular cell volume (MCV) ≤ 82 fl. Body iron stores (mg) were estimated with the following formula: (baseline Hct + NID Hct/2) × blood volume removed × 1 mg/mL. Baseline measurements were repeated at 30–45 days after discontinuation of phlebotomies. A preliminary study23 indicated that, within such time span, Hct entirely returns to baseline levels. The potential confounding effect of changes of blood viscosity on blood pressure regulation were therefore avoided. Six months later, patients were studied again (Fe replenishment control phase). Throughout the study, an effort was made to avoid changes in medication and lifestyle habits.

Statistical Analyses Results are expressed as means ± SEM. Frequency distributions were estimated for each variable. Mean values at NID were compared with baseline and 6-month post-NID results by paired (two-tailed) Student’s t test or the Wilcoxon-matched pairs test for normally distributed and nonparametric correlated variables, respec- tively. Nonparametric variables were serum fructosamine, ferritin, triglyceride, and post-OGTT insulin concentrations. Associations among serum indexes of Fe status and size of Fe stores were estimated by calculating Pearson’s correlation coefficients. Statistical calculation was performed with a commercial software (Statsoft-Inc., Tulsa, OK) for the MacIntosh (iMAC, Apple Computers, Cupertino, CA).

RESULTS

Demographic and clinical variables of the 31 study patients are shown in TABLE 1. No changes in body weight were observed. Mean weight was 90 ± 4 kg at baseline, 90 ± 4 kg at NID, and 88 ± 4 kg at 6 months after NID (p = NS). Phlebotomy lowered both SFC and Fe saturation to target values (TABLE 2), while MCV fell from 88 ± 1 fl to 81 ± 1 fl (p < 0.001). A cumulative, average blood volume FACCHINI & SAYLOR: IRON DEPLETION 345

TABLE 1. Demographic and clinical characteristics N (M/F) 31 (21/10) Age (years) 51 ± 4 Weight (kg) 90 ± 4 BMI (kg/m2)27 ± 2 Type-2 DM (%) 70 (21/31) HTN (%) 78 (24/31) Both (%) 52 (16/31) Duration of type-2 DM (years) 4 ± 1 Duration of HTN (years) 9 ± 2 Smoker status (%) 12 (4/31) Type-2 DM on OHA (%) 82 (18/22) HTN on ≥2 drugs (%) 75 (18/24)

NOTE: Type-2 DM = type-2 diabetes mellitus; HTN = essential hypertension; OHA = oral hypoglycemic agent.

TABLE 2. Size of body Fe stores (mg of Fe removed) and serum parameters of Fe status at baseline, at near-iron deficiency (NID), and 6 months after phlebotomies were discontinued Variable Baseline NID 6 months p Fe stores 1535 ± 122 112 ± 8* 360 ± 64* <0.001 Ferritin (µg/L) 272 ± 32 14 ± 145 ± 8 <0.001 Iron (µmol/L) 17 ± 0.9 8 ± 0.5 15 ± 1 <0.001 TIBC (g/L) 3.2 ± 0.08 4.2 ± 0.08 3.7 ± 0.08 <0.001 Fe-sat 38 ± 210 ± 123 ± 2 <0.001 MCV (fl) 88 ± 181 ± 185 ± 1 <0.001 Hct (%) 42 ± 141 ± 142 ± 1NS

NOTE: p values—baseline vs. NID. TIBC = transferrin; Fe-sat = iron saturation; MCV = mean corpuscular volume of red blood cells; Hct = hematocrit; * = estimated from SFC (references 24 and 25). of 3646 ± 155 mL was removed, corresponding to 1.5 ± 0.1 g of Fe. Stored Fe poorly correlated with SFC (r = 0.4), explaining only about 20% of the SFC variance. This information is shown in TABLE 3. From TABLE 3, it can also be seen that neither Fe saturation nor MCV correlated with Fe removed. At NID, statistically significant hemodynamic and metabolic changes occurred (TABLES 4 and 5; FIGURE 1). Ambulatory blood pressure recordings were an average of 20 ± 2 readings/session- patient vs. 21 ± 2 vs. 18 ± 1 at baseline, NID, and 6 months after NID, respectively. When average ambulatory blood pressure readings were considered (TABLE 4), both SBP and DBP were 10% lower at NID than at baseline (p < 0.001). Six months later, SBP returned to baseline values, while DBP was still lower (p < 0.02) than at baseline. TABLE 5 and FIGURE 1 show that chronic (HbA1c, fructosamine) and acute 346 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 3. Correlation coefficients among Fe stores and serum parameters of Fe status Variable r p Ferritin 0.40 <0.05 TIBC −0.36 <0.05 Iron 0.08 NS Fe-saturation 0.12 NS MCV 0.07 NS Hct 0.14 NS

NOTE: r = Pearson’s correlation coefficients; TIBC = transferrin; Fe-saturation = serum iron/ TIBC; MCV = mean corpuscular volume; Hct = hematocrit.

TABLE 4. Hemodynamic changes at NID and 6 months later Variable Baseline NID 6 months SBP (mmHg) 150 ± 4148 ± 4156 ± 5 DBP (mmHg) 85 ± 283 ± 287 ± 3 AMBU SBP 160 ± 4 146 ± 5* 156 ± 4 AMBU DBP 91 ± 380 ± 3* 87 ± 3† AMBU HR (BPM) 79 ± 274 ± 2† 81 ± 2

NOTE: Significances—*p < 0.001; †p < 0.02. SBP: systolic blood pressure; DBP: diastolic blood pressure; AMBU: mean ambulatory blood pressure and heart rate (HR) recordings were calculated from an average of 20 ± 2 vs. 21 ± 2 vs. 18 ± 1 readings/session-patient at baseline, at NID, and 6 months after NID, respectively.

TABLE 5. Metabolic changes at NID and 6 months later Variable Baseline NID 6 months FAGlu (mmol/L) 8.7 ± 0.5 7.5 ± 0.4* 8.8 ± 0.5 FAIns (pmol/L) 230 ± 27 165 ± 20† 181 ± 19 HbA1c (%) 8.0 ± 0.3 6.9 ± 0.2* 7.7 ± 0.4 Fructos. (µmol/L) 300 ± 12 268 ± 8* 292 ± 13 TC (mmol/L) 5.6 ± 0.2 4.9 ± 0.2* 5.3 ± 0.2 TG (mmol/L) 1.87 ± 0.1 1.54 ± 0.1* 1.86 ± 0.2 LDL-C (mmol/L) 3.46 ± 0.2 2.80 ± 0.2* 3.30 ± 0.2 HDL-C (mmol/L) 1.09 ± 0.05 1.29 ± 0.05* 1.20 ± 0.05† TC/HDL 5.2 ± 0.2 4.0 ± 0.2* 4.7 ± 0.2‡ Homocysteine (µmol/L) 9 ± 19 ± 1 9 ± 1 Fibrinogen (g/L) 3.4 ± 0.1 2.9 ± 0.1* 3.1 ± 0.1

NOTE: Significances—*p < 0.001; †p < 0.01; ‡p < 0.02. FAGlu = fasting glucose; FAIns = fasting insulin; Fructos. = fructosamine; TC = total cholesterol; TG = ; LDL-C = low-density lipoprotein cholesterol; HDL-C = high-density lipoprotein cholesterol. FACCHINI & SAYLOR: IRON DEPLETION 347

FIGURE 1. (Top) Plasma glucose concentrations during a with measurements at baseline and 60, 120, and 180 min. Paired values at baseline, NID, and 6 months post-NID are illustrated. (Bottom) Plasma insulin concentrations during a glucose tolerance test with measurements at baseline and 60, 120, and 180 min. Paired values at baseline, NID, and 6 months post-NID are illustrated. 348 ANNALS NEW YORK ACADEMY OF SCIENCES

(OGTT glucose and insulin) glucoregulatory indexes were 10–30% lower at NID than at baseline (p < 0.001 to p < 0.01). When comparing baseline with NID, the reduction of plasma insulin values was more pronounced at 120 min (from 984 ± 72 to 675 ± 50 pmol/L; p < 0.01) and 180 min (from 818 ± 70 to 415 ± 31 pmol/L; p < 0.001) than at peak levels (60 min) (862 ± 62 vs. 969 ± 72 pmol/L; p = NS). Reduc- tions of glucose and insulin concentrations were paralleled by changes in total cho- lesterol (−12%; p < 0.001), triglyceride (−20%; p < 0.001), LDL-cholesterol (−20%; p < 0.001), TC/HDL (−24%; p < 0.001), and fibrinogen (−15%; p < 0.001), while plasma HDL-cholesterol was increased (+18%; p < 0.001). Homocysteine concen- tration was unchanged. Six months (199 ± 6 days) after interruption of phlebotomy, most ASCVD risk factors returned to baseline values (TABLE 5), with the exception of fasting insulin (−21%; p < 0.02), TC/HDL (−10%; p < 0.02), and HDL-C (+10%; p < 0.01).

DISCUSSION

To the aim of quantifying body Fe stores, quantitative phlebotomy remains the standard of reference.22,24,25 In the current investigation, 31 CHO-intolerant patients underwent quantitative phlebotomy and their body Fe stores were normal. Former liver biopsy studies came to the same conclusion,26 while Moirand et al. reported Fe stores of 2.5 g in individuals with clinical evidence of insulin resistance.27 The much greater SFC (>500 µg/L vs. 272 µg/L) and prevalence of males (M:F = 12:1 vs. 2:1) probably accounted for the additional 1 g of Fe found in that study. Moreover, those patients were selected on the basis of increased SFC, while selection in the present study strictly relied on different criteria. Therefore, rather than discordant, the present results integrate those from Moirand et al. and indicate an important inter- relationship among Fe and CHO-intolerance, independent of hemochromatosis, and detectable even at lower values of the storage Fe frequency distribution curve. Thus, CHO-intolerant patients have normal or mildly elevated body Fe stores and use of SFC would have overestimated their iron burden of 40–70%.24,25 Hallberg et al., by directly measuring body Fe in normal subjects, came to similar conclusions and sug- gested that factors other than Fe storage may become more important determinants of SFC above 70 µg/L.6 In this context, it was demonstrated that insulin-resistant individuals often present elevation of inflammatory indexes, such as fibrinogen, WBC count, and C-reactive protein.28 Phagocyte-mediated, Fe-catalyzed oxidant damage to the endothelium is presumably involved in atherogenesis, and endothelial ferritin synthesis is an important cytoprotective antioxidant stratagem.29 Thus, there is the possibility that the hyperferritinemia noted in individuals with the Metabolic Syndrome X30,31 reflects ongoing oxidant-induced endovascular inflammation rather than increased body Fe stores. Other abnormalities commonly found in individuals with the Metabolic Syndrome X (or insulin resistance) are hyperfibrinogenemia,28,32 hyper-LDL cholesterolemia,16 hypertriglyceridemia, hypertension, and low HDL- cholesterolemia, as well as relative and marked hyperinsulinemia after CHO intake.13 These individuals are at greater risk of developing type-2 diabetes and all together, or in various combinations, these abnormalities make them prone to ASCVD. In the current investigation, all of these abnormalities, which are known to cluster13,16,28,30,31 in insulin-resistant individuals with the exception of plasma FACCHINI & SAYLOR: IRON DEPLETION 349 homocysteine concentration,33 improved with depletion of Fe stores to NID. In par- ticular, both plasma glucose and insulin responses to oral glucose loading were about 30% lower than at baseline. This finding indicates a state of reduced insulin resistance34 and that one of NID’s most important effects was to enhance insulin- stimulated glucose disposal. Considerable evidence, in vitro and in vivo, supports this notion. Potashnick et al. demonstrated upregulation of GLUT-1 in cultured myo- cytes incubated with iron chelators with a 2-fold increase of glucose uptake.35 Glu- cose transport also doubled in rats36 and veal calves37 made Fe-deficient and anemic by Fe-poor diets, while venesection to NID increased ~50% glucose disposal in lean, normal humans.38 Thus, regardless of the method used to induce Fe depletion (bleeding, Fe-chelation, or selective Fe dietary exclusion) the same effect was observed: a 50–200% increase of glucose . The fact that this effect occurred with chelators and with normal hematocrits, as in the present study, indi- cates that Fe, rather than anemia, is the glucose tolerance (GT) factor. However, Fe depletion might enhance glucose metabolism indirectly, for example, by stimulating norepinephrine turnover.39 Regardless of the specific mechanism involved, the present study demonstrates that the insulin-sparing effect of NID also occurs in CHO-intolerant humans and likely influenced the significant improvement of all Syndrome X–related ASCVD risk factors. In fact, similar improvements are known to happen with any other insulin-sparing agent, for example, physical activity, calorie restriction, metformin, or thiazolidinediones. However, such changes were largely abolished after 6 months of Fe repletion, despite the fact that storage Fe remained below baseline levels. Thus, for individuals at high risk for ASCVD, NID seems a more favorable metabolic state than Fe sufficiency. This view is congruous with the cardiovascular protection of premenopausal, menstruating females who are often near-iron-deficient10 as well. The fact that hysterectomy eliminated such pro- tection,12 while recent controlled trials of estrogen replacement had neutral or nega- tive impact on ASCVD,11 lends further credence to the notion that menstrual bleeding, not estrogen, is the protective factor. Obviously, phlebotomy removes many elements besides Fe. Could substrates other than Fe have modified glucose homeostasis? Such possibility appears unlikely as lowering of no other metabolite or ion is known to enhance GT. On the contrary, depletion of trace elements such as Zn,40 Cu,41 and Cr42 should worsen, not enhance, GT, indicating the possibility that dietary Fe depletion may be more effective than phlebotomy. In summary, Fe stores were normal in individuals at high risk for ASCVD. Secondary to the insulin-sparing effect of NID, risk factors for ASCVD were im- proved at NID and worsened again by iron sufficiency. It is concluded that depletion of Fe to levels similar to those of premenopausal females has beneficial effect on most cardiovascular risk factors of CHO-intolerant individuals.

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