NUTRITIONAL STATUS AND LIPID PROFILES IN ACTIVE WOMEN WITH AND WITHOUT MENSTRUAL DYSFUNCTION RB Biegler, J Chang, DL Finders, EE Sperber, LC Kam, CP Guebels, MM Manore, FACSM.Department of Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR.

Exercise-induced menstrual dysfunction (ExMD) is prevalent (6-79%) in active women and may result from low energy availability. Unfavorable lipid profiles have been reported in women with ExMD; low energy intake may also increase risk for poor nutritional status. PURPOSE: To compare nutritional status (diet, blood) and lipid profiles in active women with ExMD 1) before and after a 6-mo intervention that provided a daily fortified CHO-PRO supplement (360 kcal/d; 300 mg/d calcium [Ca]) and 2) to Eumenorrheic controls (EU). METHODS: Menstrual status was confirmed by measuring reproductive hormones. In the ExMD group (n=8; age=23±3y, VO2max=49±6 mL/kg/min, body fat= 22±5%), energy intake and expenditure were assessed at pre- (0-mo) and post-intervention (6-mo) using 7-d weighed food and physical activity records. EU athletes (n=10; age= 24±5y, VO2max=51±5 mL/kg/min, body fat=23±4%) were only measured at 0-mo. RESULTS: For ExMD women, the addition of a CHO-PRO supplement to the diet increased Ca intake (pre=1320±571 mg/d, post=1725±555 mg/d, p=0.02). Dietary cholesterol (chol) (pre=193±130g/d; post=339±240 g/d, p=0.04) also increased, but this was not due to the CHO-PRO drink (chol=15mg/325 ml). At 6-mo, Ca intake was significantly higher in ExMD vs. EU (1211±385 mg/d, p=0.04), but not at 0-mo. Vitamin/mineral supplement use was more prevalent in EU (50%) compared to ExMD (13-38%, range during intervention). At 6-mo, mean Total chol (188 mg/dL) and LDL-chol (103mg/dL) were higher in ExMD compared to EU (Total chol=154 mg/dL; LDL-chol=84 mg/dL; p<0.05), yet mean cardiac risk was similar (p>0.05; ExMD=2.6; EU=3.0) due to higher HDL-chol levels in ExMD (71 mg/dl) vs. EU (53 mg/dL). In the ExMD group, 25-38% had low iron status during the intervention vs. 20% of EU (0-mo). No other differences in blood micronutrient status were observed. CONCLUSION: Although, Total chol and LDL-chol were higher in ExMD vs. EU, overall both groups had low cardiac risk. Ca intake was higher in ExMD group due to the added Ca in the CHO-PRO supplement. No other group differences in diet or blood profiles were observed. In conclusion, ExMD may negatively increase some blood lipid parameters but this did not translate into increased cardiac risk.

ABSTRACT

INTRODUCTION

PURPOSE

METHODS

Energy/Nutrients CHO-PRO Supplement (325 mL)Low Fat Chocolate

Milk (325 mL)

Energy (kcal) 360 216Carbohydrate (g) 54 36Protein (g) 20 11Fat (g) 8 3Calcium (% DV) 30 39Vitamin D (% DV) 25 69Iron (% DV) 10 5Folic Acid (% DV) - 4Vitamin B6 (% DV) 20 11Vitamin B12 (% DV) 20 49

REFERENCES

ACKNOWLEDGEMENTS

DISCUSSION AND CONCLUSION

RESULTS: ExMD 0-mo vs. 6-mo RESULTS: ExMD vs. EU

DescriptionMean (SD)

ExMD (n=8) EU (n=10)0-mo 6-mo 0-mo*

Age (y) 22.6 (3.3) N/A 24.1 (4.6)Age at menarche (y) 13.5 (2.0) N/A 12.7 (1.3)

Weight (kg) 62.4 (7.8) 64.0 (8.0)a 66.8 (9.3)

BMI (kg/m2) 22.3 (2.5) 22.9 (2.5)a 23.2 (2.8)FFM (kg) 48.5 (4.6) 48.4 (4.8) 51.0 (5.0)

Body Fat (%) 22.0 (4.7) 24.1 (3.9)a 23.2 (4.4)Training Volume (h/wk) 7.4 (3.2) 7.1 (3.4) 7.4 (3.6)VO2max (mL/kg/min) 49.0 (5.8) 49.3 (6.0) 50.6 (5.2)EB (kcal/d) -510 (361) -44 (707) -171 (459) #

EA (kcal/kg FFM/d) 37 (10) 45(15) 38 (10) #

The prevalence of exercise-induced menstrual dysfunction (ExMD) is high (6-79%) in active women (Beals & Hill, 2006; Beals & Manore, 2002). Highest rates have been reported in aesthetic (30-43%) and endurance (27-32%) sports, particularly those where a lean physique may offer a competitive advantage (Beals & Hill, 2006; Beals & Manore, 2002; Nichols, Sanborn, & Essery, 2007; Torstveit & Sundgot-Borgen, 2005). Some studies (Friday, Drinkwater, Bruemmer, Chestnut, & Chait, 1993; Rickenlund, Eriksson, Schenck-Gustafsson, &Hirchberg, 2005) have reported an association between ExMD and unfavorable lipid profiles, while others (Hinton, Rector, Peppers, Imhoff, & Hillman, 2006; Hoch, et al., 2003) report no differences in lipid profiles between ExMD and Eumenorheic athletes (EU). Elevated blood lipids may increase cardiac risk: ratio of Total chol (CHOL) to high-density lipoprotein chol (HDL-chol). In addition to increased cardiac risk, women with ExMD may be at risk for poor nutritional status due to low energy intake. Dietary intake of micronutrients, notably B-vitamins (folate, B-6, B-12), iron, zinc, and bone building nutrients (calcium, magnesium, and vitamin D), may be insufficient in this population (Manore, 1999, 2000, 2002; Woolf & Manore, 2006). Exercise may increase the need for some of these micronutrients (Manore, 2000, 2002). Lastly, low iron status is also prevalent in active women (~35% of population) (Brownlie, Utermohlen, Hinton, & Haas, 2004; Dubnov & Constantini, 2004; Hinton, Giordano, Brownlie, & Haas, 2000; Sinclair & Hinton, 2005; Zhu &Haas, 1998). Inadequate dietary iron intake, diets with low iron bioavailability, and excessive iron loss may contribute to the high incidence of iron deficiency in this population (Manore, 2002).

The primary purpose of this study was to:1) Determine nutritional status, lipid profiles, and iron status of active women with ExMD before and after a 6-mo diet intervention that provided a daily fortified CHO-PRO supplement (360 kcal/d; 20 g/d PRO, 54g/d CHO, 300 mg/d calcium [Ca]).2) Compare nutritional status, lipid profiles, and iron status between women with ExMD and active Eumenorrheic controls (EU).

Endurance-trained women (n=18) were recruited from the Willamette Valley and divided into two groups based on menstrual status. Participation criteria included being 18-35y, exercising regularly (minimum 7 h/wk), no use of oral contraceptives or hormone therapy for the last 6 months, and a score <14 on the Eating Disorder Inventory (EDI-2) assessment (Garner & Olmstead, 1984). Active women with ExMD participated in a 6-mo diet intervention, which provided a daily CHO-PRO supplement (360 kcal/d; 20 g/d PRO, 54 g/d CHO, 300 mg/d Ca). See Table 1 for a comparison of the CHO-PRO supplement to low fat chocolate milk. Self-reported menstrual dysfunction was confirmed by measuring reproductive hormones in the blood (estradiol, progesterone, luteinizing hormone, follicle stimulating hormone, prolactin) and determination of ovulation status (ClearBlue ® Easy Fertility Monitor). ExMD participants (n=8) were assessed at baseline (0-mo) and post-intervention (6-mo) for energy intake and expenditure, body composition, VO2max, and blood parameters (iron status, lipid profile, folate, vitamin B-12, vitamin D); active EU controls (n=10) completed the same measurements at 0-mo only. Cardiac risk was reported as the ratio of Total cholesterol (CHOL)/ high-density lipoprotein cholesterol (HDL-chol). Body composition was determined by dual-energy x-ray absorptiometry (DXA) (Hologic QDR-4500 Elite A Waltham, MA) and a continuous graded exercise test was used to assess VO2max using indirect calorimetry (TrueOne 2400; ParvoMedics Metabolic Cart, Sandy, UT). Dietary energy (EI) and nutrient intake were quantified using 7-d weighed food records; we screened for under-reporters using the Goldberg cut-off (1.35 x Basal Metabolic Rate [BMR]) (Goldberg, et al. 1991). One EU participant was dropped from the dietary intake data due to under-reporting. Physical activity logs were kept on the same 7-d and allowed for estimations of total and exercise energy expenditure (TEE and EEE, respectively). All records were analyzed using a computerized diet and activity analysis program (Food Processor SQL version 9.91, 2006; ESHA Research).

Energy balance (EB; EB= EI-TEE) and energy availability (EA; EA=EI-EEE) were calculated from the analyzed records, adjusting for resting metabolic rate (RMR) (measured 2x using indirect calorimetry) and running energy expenditure (REE) (typical training speeds; indirect calorimetry). For the ExMD group, one-sided paired t-tests were performed to determine changes over time (0-mo vs 6-mo) for parameters we hypothesized would improve (EI, EB, EA, body weight, and micronutrients provided by the supplement); all other ExMD comparisons over time were performed using 2-sided paired t-tests. Between-group comparisons (ExMD 0-mo vs. EU, ExMD 6-mo vs. EU) were made using two-sided unpaired t-tests. Data were summarized as means and standard deviations. Statistical significance was set at p<0.05.

DescriptionMean (SD)

Recommended IntakeExMD (n=8) EU (n=9)#

0-mo 6-mo 0-mo*

Energy intake (kcal/d) 2312 (325) 2694 (541)a 2430 (524) variable

Carbohydrate (g/kg/d) 5.0 (1.2) 5.4 (1.0) 4.6 (1.0) ≥5 g/kg/d

Carbohydrate (%) 53 (7) 51 (6) 50 (5) 55-65%

Protein (g/kg/d) 1.4 (0.2) 1.8 (0.5) 1.3 (0.3)c endurance:1.2-1.4 g/kg/d strength:1.6-1.7 g/kg/d

Protein (%) 15 (4) 17 (2)a 15 (3) 10-35%Fat (g/kg/d) 1.2 (0.3) 1.5 (0.6) 1.4 (0.4) N/AFat (%) 29 (3) 30 (5) 34 (5)b 20-35%Saturated fat (%) 8.3 (2.5) 8.8 (2.0) 10.8 (2.2)b <10%

Cholesterol (mg/d) 193 (130) 339 (240)a 259 (132) <300 mg/d

•The diet intervention, which provided an additional 360 kcal/d, increased mean body weight and BMI (p=0.03); increases in body weight were primarily as fat mass (+2.1±2.1% body fat; p=0.03) (see Table 2).

•Mean dietary protein intake [% of energy intake (EI)] increased from 0-mo to 6-mo (p<0.05). See Table 3a for a summary of dietary energy and macronutrient intake compared to recommendations.

•At 0-mo, mean dietary calcium intake (1320 mg/d) was below the recommended amount for active women with ExMD (1500 mg/d) (Nattiv, et al., 2007); however, significant improvements were observed with the addition of the CHO-PRO supplement by 6-mo (1725 mg/d; p=0.02). Intakes of folate, B-6, B-12, iron, magnesium, phosphorus, and zinc were similar before and after the intervention (p>0.05). Mean dietary intake of vitamin D was below the recommendation at both time points (0-mo and 6-mo), but sun exposure was not measured during the study. Mean magnesium intake was < Recommended Daily Allowance (RDA) at 0-mo only (Table 3b).

•No changes were observed in blood lipids or cardiac risk from 0-mo to 6-mo (p<0.05). As shown in Figure 1, triglycerides (TG), Total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-chol), low-density lipoprotein cholesterol (LDL-chol), and very low density lipoprotein cholesterol (VLDL-chol) were all within the normal reference ranges.

•Serum folate and B-12 did not change due to the intervention (p>0.05). One participant that was taking a B-12 supplement (100mcg/d) at 0-mo had serum B-12 values above the reference range and was advised to discontinue taking the supplement.

•Iron status remained similar pre- and post-intervention (p>0.05). At 0-mo, one participant was in iron depletion (low ferritin) and another had iron deficiency anemia (low serum iron, high TIBC, low % saturation, low ferritin). By 6-mo, the individual with iron depletion remained depleted and the second participant improved her iron status to iron deficiency without anemia. One participant became iron deficient without anemia (Table 4).

•All 8 participants with ExMD resumed menses over the 6-mo diet intervention (mean time to first menses = 2.63 mo, range 1-7 mo).

•Women with ExMD and active EU controls were similar in age, age at menarche, body composition, training volume, and VO2max (p>0.05). Although EB (kcal/d) was higher for EU than ExMD at 0-mo, differences did not reach significance (p=0.06). There were no significant between-group differences for EA (p>0.05). See Table 2 for participant characteristics.

•Dietary protein intake of EU (1.3 g/kg/d) was adequate, but significantly lower than ExMD at 6-mo (1.8g/kg/d; p=0.04). The percent of energy from fat was higher in EU (34%) than ExMD at both time points (0-mo: 29%; 6-mo: 30%); although, only statistically significant at 0-mo (p=0.045). Mean intakes of carbohydrate were similar between ExMD and EU (p>0.05); however, EU (4.6g/kg/d) were below the recommendation of 5 g/kg/d for endurance female athletes (see Table 3a).

•Dietary intake of calcium was lower in EU than ExMD participants at 6-mo (p=0.04) (see Table 3b). Intakes of folate, B-6, and B-12 were similar between groups and met recommendations; however, mean vitamin D intake was inadequate (<600 IU/d) for both groups. Mineral intakes were adequate and comparable between groups (p>0.05).

•CHOL and HDL-chol were lower in EU than ExMD at both time points (p<0.05). LDL-chol was similar between EU and ExMD at 0-mo (p=0.15), but was significantly lower than ExMD at 6-mo (p=0.04). There were no differences in cardiac risk between the groups (p>0.05). See Figure 1 for more details.

•Blood levels of folate, vitamin B-12, and vitamin D were similar in EU vs. ExMD (0-mo and 6-mo, p>0.05).

•There were no significant between-group differences in blood iron status parameters (p>0.05) (see Figure 2). Two EU participant were classified as iron depleted (low ferritin), compared to 2 ExMD participants with poor iron status at 0-mo (n=1 iron depletion, n=1 iron deficiency anemia) and 3 ExMD participants at 6-mo (n=1 iron depletion, n=2 iron deficiency without anemia).

Although CHOL and LDL-chol were significantly higher in ExMD participants compared to EU controls (Figure 1), both groups had a low cardiac risk. Overall, cardiac risk ranged from 2.5 -3.0, well below the normal range of 3.7-5.6. In addition, mean blood lipids were within recommended ranges. Calcium was the only dietary micronutrient that significantly improved with the 6-mo intervention (ExMD); the increase in calcium was largely due to the Ca (300 mg/d) in the CHO-PRO supplement. Other bone-related micronutrients (Vitamin D, magnesium, phosphorus) were not significantly different over the intervention and between groups. Poor iron status was observed in a small percentage of our participants, but overall status was lower than reported for active women. Eighty-two percent of our participants consumed at or above the RDA for iron (18 mg/d). All macronutrients intakes were within recommended ranges. In conclusion, ExMD may negatively affect some blood lipid parameters compared to EU; however, this does not translate into an increased cardiac risk.

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The authors would like to acknowledge the Gatorade Sports Science Institute for funding this study. We are also grateful for support from the OSU College of Health and Human Sciences and USDA Training Grant.

ExMD, exercise-induced menstrual dysfunction; EU, eumenorrheic endurance-trained

control. BMI, body mass index; FFM, fat free mass (DXA); VO2max, maximal aerobic

capacity (indirect calorimetry). %, percent of total energy intake from 7-d diet records.

EB, energy balance (EB= energy intake [EI]- total energy expenditure [TEE])

EI, total dietary energy intake from analysis of 7-d weighed diet records

TEE, total energy expenditure from 7-d activity logs adjusted for measured RMR and

running energy expenditure.

RMR, resting metabolic rate (measured 2x at each time point; indirect calorimetry).

EA, energy availability (EA= energy intake [EI] – exercise energy expenditure [EEE]).

Training volume, all minutes of exercise (planned + unplanned) >4.0 METs from 7-d activity logs.

EEE, all kcals expended during exercise (planned + unplanned) >4.0 METs from 7-d activity logs.

*EU were measured at baseline (0-mo) only and compared to ExMD at 0-mo and 6-mo.#one EU participant was left out due to under-reporting (Goldberg cut-off: 1.35 x basal metabolic rate).(+)The Female Athlete Triad Position Stand (Nattiv, et al., 2007) recommends that active

women with amenorrhea consume1500mg/d of Calcium. aSignificant difference between ExMD at 0-mo vs. 6-mo (p<0.05). bSignificant difference between EU vs. ExMD at 0-mo (p<0.05).cSignificant difference between EU vs. ExMD at 6-mo (p<0.05).

Description

Mean (SD)Recommended

IntakeExMD (n=8) EU (n=9)#

0-mo 6-mo 0-mo*Vitamin B-6 (mg/d) 3.6 (2.8) 2.6 (1.0) 3.4 (2.3) 1.3Folate (mcg/d) 532 (468) 426 (236) 449 (207) 400Vitamin B-12 (mcg/d) 14 (25) 7 (4) 8 (5) 2.4Iron (mg/d) 29 (15) 22 (5) 24 (9) 18Vitamin D (IU/d) 379 (321) 383 (316) 385 (314) 600Calcium (mg/d) 1320 (571) 1725 (555)a 1211 (385)c 1000(+)

Magnesium (mg/d) 288 (115) 330 (69) 365 (194) 310Phosphorus (mg/d) 912 (383) 1034 (386) 1098 (471) 700Zinc (mg/d) 13 (8) 16 (5) 14 (6) 8

*ExMD (0-mo) vs. EU; p<0.05.†ExMD (6-mo) vs. EU; p<0.05. ExMD, exercise-induced menstrual dysfunction (n=8); EU, eumenorrheic active controls (n=10). TG, triglycerides; CHOL, Total cholesterol; cardiac risk= CHOL/HDL-chol.

Figure 2. Iron deficiency panel.Fe, iron; TIBC, total iron-binding capacity; saturation %, transferrin carrier; ferritin, iron storage. ExMD, exercise-induced menstrual dysfunction (n=8); EU, active eumenorrheic control (n=10).

Figure 1. Lipid profile

Table 2. Participant characteristics.

Table 3a. Dietary energy and macronutrient intake.

Table 3b. Dietary micronutrient intake.

Table 1. Comparison of CHO-PRO supplement to low fat chocolate milk.

% DV =% of Daily Values ‡ADA-ACSM Position Stand on Nutrition and Athletic Performance (2009); Manore (2002); Dietary Guidelines for Americans (2010).

See further footnotes below.

See footnotes on right.

See further footnotes on right..

‡ Dietary Reference Intakes for Calcium, Phosphorous, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline (1998); and Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron,Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001); and Dietary Reference Intakes for Calcium and Vitamin D (2011). These reports may be accessed via www.nap.edu.

Footnotes for Tables 2-3.

Stage ExMD (0-mo)

ExMD (6-mo)

EU (0-mo)

Stage I: Iron depletion 1 1 2

Stage II: Iron deficiency without anemia

0 2 0

Stage III: Iron deficiency with anemia

1 0 0

ExMD, exercise-induced menstrual dysfunction (n=8); EU, Eumenorrheic active control (n=10); Iron depletion= low ferritin; Iron deficiency without anemia= low serum iron, low % saturation; Iron deficiency anemia= low serum iron, high TIBC, low % saturation, low ferritin.

Table 4. Comparison of iron status.

+