Plasma, Urinary, Erythrocyte, and Platelet Concentrations of Manganese and Molybdenum in Football Players: Differences between Sexes and during the Season

Abstract

Physical activity induces modifications in the concentrations of trace mineral elements. However, studies exploring sex-related differences in manganese (Mn) and molybdenum (Mo) levels among athletes are scarce. Mn and Mo are essentials metals required for a variety of metabolic functions, including those involved in normal human development, the activation of certain metalloenzymes, energy metabolism, and immune system function. They are important cofactors for a variety of enzymes, including those involved in neurotransmitter synthesis and metabolism. The presence of molybdenum (Mo) is essential for several enzymes, including xanthine oxidase (XO), aldehyde oxidase, sulfite oxidase (SO), and the mitochondrial amidoxime reductase component (mARC). This study aimed to: (a) analyse changes in plasma, urine, erythrocyte, and platelet Mn and Mo concentrations throughout a competitive season in men’s and women’s football players, and (b) investigate sex-based discrepancies. A total of 46 football players (22 men: age; 20.62 ± 2.66 years; height; 1.76 ± 0.061 m; weight; 71.50 ± 5.93 kg, and 24 women: age; 23.21 ± 4.11 years; height; 1.65 ± 0.06 m; weight; 59.58 ± 7.17 kg) participated in this study. Three assessments were conducted throughout the competitive season. Data were collected on anthropometry, body composition, nutritional intake, physical fitness, female hormones, haematology, and the determination of Mn and Mo in different biological compartments. Regarding Mn, significant sex differences were observed in plasma, urine, and erythrocyte concentrations (p < 0.05). Moreover, significant variations were observed throughout the season in all analysed biological compartments (p < 0.05). Regarding Mo, significant sex differences were reported in plasma concentrations (p < 0.05). Similarly, there were variations throughout the season in all analysed biological compartments (p < 0.05). Plasma, urine, erythrocyte, and platelet Mn and Mo concentrations could change during a competitive season in football players. On the other hand, sex differences could exist in plasma, urine, and erythrocyte Mn concentrations in football players.

1. Introduction

Trace mineral elements (TMEs) are involved in hundreds of biological processes relevant to exercise and sports performance, such as energy storage/utilisation, protein metabolism, inflammation, oxygen transport, heart rate, bone metabolism, and immune function [1,2]. Under conditions of high metabolic demand, inadequate circulating and cellular TME levels can compromise optimal physiological performance [1,3]. While extensive research exists on trace mineral elements (TMEs) such as iron (Fe), copper (Cu), selenium (Se), and zinc (Zn), other TMEs like manganese (Mn) and molybdenum (Mo) have received less attention in the context of sports science.

Manganese (Mn) is a metal belonging to group 7 of the periodic table, and ranks as the twelfth most abundant element in the Earth’s crust [4]. Data on average Mn concentrations in human fluids exhibit considerable variation. In blood, Mn concentrations range from 0.008 to 0.05 mg/L, while in breast milk, they range from 0.0032 to 0.12 mg/L. The highest Mn content in the body is found in the bones of terrestrial mammals (up to 10 mg/kg) and in the hard tissues of aquatic animals [5].

Manganese (Mn) is an essential nutrient required for a variety of metabolic functions, including those involved in normal human development, the activation of certain metalloenzymes, energy metabolism, immune system function, nervous system function, reproductive hormone function, and in antioxidant enzymes that protect cells from damage due to free radicals [4]. Manganese (Mn) also plays a crucial role in regulating cellular energy, connective tissue and bone growth, and blood coagulation. Additionally, Mn is an important cofactor for a variety of enzymes, including those involved in neurotransmitter synthesis and metabolism [6].

Mn absorption decreases as dietary intake increases. On the other hand, Mn absorption increases with low Mn status. Thus, variable absorption is apparently a significant factor in the regulation of Mn homeostasis, to which its excretion contributes. Mn is found predominantly in the mitochondria and, therefore, the liver, kidney, and pancreas have relatively high Mn concentrations. On the other hand, Mn is present in extremely low concentrations in human plasma and urine [7]. Gastrointestinal Mn absorption involves the main intestinal iron transporters, such as Divalent Metal Transporter 1 (DMT1) (Meltzer et al., 2010). The activity of this transporter increases when Fe stores are low, which would explain the increased Mn absorption under conditions of anemia or Fe deficiency [7].

Manganese superoxide dismutase (MnSOD) is the primary antioxidant enzyme in mitochondria [3]. Physiological stress induced by exercise training enhances MnSOD activity in the myocardium, providing protection against ischaemia-reperfusion-induced arrhythmias and infarctions [8]. Manganese (Mn) may play a significant role in protecting against oxidative damage induced by high-intensity training and promoting workout recovery [9]. Manganese (Mn) is also essential for the activity of both pyruvate carboxylase and phosphoenolpyruvate carboxykinase, enzymes that play crucial roles in the gluconeogenic pathway by catalysing the first rate-limiting step [10].

Molybdenum (Mo) is a metal belonging to group 6 of the periodic table and is found in low concentrations in living organisms, with the highest concentrations being found in the liver, pancreas, and small intestine [11]. Mo is not biologically active, as it acts as a cofactor for a limited number of enzymes in humans by forming the molybdenum cofactor. The presence of molybdenum (Mo) is essential for several enzymes, including xanthine oxidase (XO), aldehyde oxidase, sulfite oxidase (SO), and the mitochondrial amidoxime reductase component (mARC). SO is believed to be the most important molybdoenzyme for health. SO localizes to the mitochondrial intermembrane space, where it catalyses the oxidation of sulfite to sulfate. XDH and XO play important roles in purine catabolism and catalyse the last two oxidative reactions that convert hypoxanthine to xanthine and xanthine to uric acid, which is eventually excreted in the urine. mARC is the most recently discovered molybdoenzyme in humans, and two different isoforms have been described. mARC proteins are involved in drug metabolism, as they were found to metabolize several N-hydroxylated compounds commonly used as prodrugs [12]. These enzymes play crucial roles in the oxidation of purines to uric acid, the metabolism of aromatic aldehydes and heterocyclic compounds, and the catabolism of sulfur-containing amino acids [12].

Mo absorption occurs rapidly in the stomach and throughout the small intestine, with the absorption rate being higher in the proximal than in the distal parts. The amount absorbed in the body depends not only on the level of Mo intake, but also on the presence of Cu and sulfate in the diet. It is believed that a high content of inorganic sulfate in the diet blocks the transport of Mo across the cell membrane, which reduces intestinal absorption and renal tubular reabsorption. The entry and exit of Mo into the circulation and tissues, especially the liver, is characterized by a rapid accumulation. MoO42- is transported to erythrocytes where it tends to bind specifically to α-2-macroglobulin [12].

In relation to physical exercise, Mn is a constituent of multiple enzymes, including mitochondrial SOD, which is fundamental in antioxidant systems. It has been proven that physical exercise stimulates the synthesis of mitochondrial SOD in order to protect the organism from free radicals generated by physical activity. It has been observed that high concentrations of Mo in plasma and urine could facilitate the formation of uric acid, considered an antioxidant substance, thus reducing the damage caused by free radicals, such as superoxide anions, generated by XO in ischaemia-reperfusion processes during high-intensity physical exercise [13,14]. The organic need to produce this enzyme in athletes may be the reason for the higher Mo concentrations, as they suggest that the enzyme protects cells against free radicals produced in muscle cells [14]. Mo is a remarkable element that possesses the ability to readily change its oxidation state, making it an ideal candidate for acting as an electron transfer agent in oxidation–reduction (redox) reactions. This unique property forms the foundation for the catalytic process of molybdenum-containing enzymes (molybdoenzymes), enabling them to facilitate the hydroxylation of various substrates using oxygen from water [15].

Trace elements (TMEs) are essential micronutrients required for various biological processes, yet their health effects can manifest differently in men and women due to variations in their kinetics and modes of action [16]. Several biological factors contribute to these sex-based disparities, including changes associated with menarche, pregnancy, lactation, and menopause, all related to estrogens [17]. In addition to reproductive function, female sex hormones are known to affect numerous cardiovascular, respiratory, thermoregulatory, and metabolic parameters. It is also known to have implications for exercise physiology, for example, through fluid retention, changes in body temperature, and energy metabolism. Fluctuations in hormones during the cycle generate a number of confounding variables that affect performance, making study design and subsequent interpretation of results difficult [17]. While some female athletes feel a decrease in their physical capacity throughout their menstrual cycle, other athletes have produced Olympic medal-winning performances during all phases of the menstrual cycle. Several studies have demonstrated the influence of the menstrual cycle on the performance of athletes [16].

Earlier findings have examined Mn concentrations in athletes, primarily analysing levels in serum, urine [18,19], and erythrocytes [20]. However, information on Mn levels in platelets is still scarce. Research suggests that Mn may influence energy production, antioxidant defense, and bone health, potentially impacting athletic performance and recovery [14,21,22]. Other studies have highlighted the importance of assessing both intracellular and extracellular concentrations of trace elements to fully evaluate their status due to potential discrepancies among biological matrices [21,23,24]. In view of the above, the objectives of this work were as follows: (a) to analyse the changes in Mn and Mo concentrations in plasma, urine, erythrocytes, and platelets during a sports season in soccer players and (b) to analyse the differences between sexes. Based on previous research [21,23,24], we hypothesised that Mn and Mo concentrations would change throughout the season. Likewise, there would be differences between sexes.

2. Materials and Methods

2.1. Study Design

The present observational research was based on a longitudinal-quasi-experimental design on two senior soccer teams (men’s and women’s). The assessments were carried out at three different times during the regular sports season: (i) first week of training, (ii) mid-season (between the end of the first and second round of the season), and (iii) last week of training of both teams, after the end of the season.

All assessments were conducted on the same week of each month, in the morning, and in the same order for all participants to avoid the effects of circadian cycles. In addition, the assessments were carried out under similar atmospheric conditions (18 to 25 °C and 45 to 55% relative humidity). On the two days prior to the assessments, the training load of both teams was reduced so that the participants performed the different assessments with the least possible fatigue.

2.2. Participants

A total of 46 soccer players divided in two groups participated in the present study: men players (n = 22; age = 20.62 ± 2.66 years; height = 1.76 ± 0.061 m; weight = 71.50 ± 5.93; experience= 14.73 ± 3.13 years) and women players (n = 24; age = 23.21 ± 4.11 years; height = 1.65 ± 0.06 m; weight = 59.58 ± 7.17 kg; experience = 14.51 ± 4.94 years). Height and weight were assessed using a wall-mounted stadiometer (Seca 220. Hamburg, Germany) and electronic digital scale (Seca 769. Hamburg, Germany), respectively. The men’s soccer players were from a team in the fifth category of Spanish soccer and the women’s soccer players from a second-category Spanish team. All the participants trained and played league matches in the same city.

The inclusion criteria for participation in the present study were: (i) to reside in the same city; (ii) healthy individuals with no significant medical history; (iii) not take medication or supplementation that included Mn or Mo during the study period or the month prior to the first evaluation; (iv) not smoke or consume drugs; (v) to have more than 5 years of experience competing in soccer; (vi) not modify nutritional and physical activity habits during the study; and (vii) not spend more than 30 days without training with the team. In addition, the women had to meet the following inclusion criteria: (viii) to have had regular menstrual cycles for at least six months before the start of the study and during the study; (ix) not suffer from problems related to the menstrual cycle; and (x) not use contraceptive methods.

All of the participants were informed of the purpose of the study and signed a consent form prior to enrolment. The protocol was reviewed and approved by the Biomedical Ethics Committee of the University of Extremadura (Cáceres, Spain) (code 135/2020) following the guidelines of the 1964 Helsinki ethical declaration.

2.3. Menstrual Cycle

Understanding the menstrual cycle is crucial, as research suggests there are fluctuations in mineral concentrations throughout the cycle [25,26]. Consequently, all assessments were conducted during the same menstrual phase whenever possible. A menstrual cycle questionnaire was completed by the participants [27]. The duration of bleeding was 4.77 ± 1.47 days and the duration of the menstrual cycle was 27.93 ± 2.78 days. All the participants had a regular menstrual cycle and had never experienced the cessation of menstruation.

2.4. Nutritional Intake

Three days before assessments, dietary intake was recorded using a food questionnaire similar to the method employed in the study of Toro-Román et al. [21]. This questionnaire aimed to capture the nutritional content of the foods consumed by participants over the three days preceding the assessments [28]. Participants recorded the types and quantities of foods consumed in a provided log throughout those three days. Researchers then used established tables [28] to convert these food entries into estimated daily consumption values of macronutrients and manganese and molybdenum (Mn and Mo).

2.5. Sample Collection

The participants were instructed to collect their first morning urine samples around 8:00–8:30 a.m. and bring them to the designated blood collection site. The urine collection process involved using the provided 100 mL containers. The coaching staff of each team received urine collection kits with containers and tubes to distribute to the players. Participants then transferred the collected urine into smaller 9 mL tubes for storage. These tubes were subsequently frozen at −80 °C until further analysis.

Following an overnight fast, blood samples (10 mL) were collected in a 15 mL tube with sodium citrate via syringe and needle. Two millilitres, placed in tubes containing a clotting agent, were used for analysis of haematology parameters using specialised equipment (Coulter Electronics LTD, Model CPA; Northwell Drive, Luton, UK). The technique was developed by an external clinical laboratory. The remaining 8 mL of blood was divided for further analysis of Mn and Mo concentrations. Four millilitres were collected in tubes containing sodium citrate, a blood thinner. Both of these citrate tubes were centrifuged at 1700 rpm for 7 min to separate the liquid portion, called plasma, from the cellular components. The top layer of the centrifuged plasma, containing platelets (cell fragments), was collected in a separate tube and centrifuged again (3000 rpm for 10 min). All of the plasma used for analysis in this medium was removed, and the remaining mucus attached to the bottom was rediluted in Milli-Q water containing the platelets for platelet analysis. This resulting platelet-rich plasma was then divided into smaller tubes and frozen at −80 °C for storage. The remaining blood (after plasma separation) was centrifuged to remove red blood cells. These red blood cells were then washed and frozen at −80 °C for storage.

2.6. Physical Fitness

Leg power was assessed using vertical jumps performed on a specialised measurement system (Optojump, Microgate) [29]. Two attempts each were performed for both the squat jump (SJ) and countermovement jump (CMJ), with only the best attempt from each included in the analysis. A 30 s rest period separated each jump. For SJ, participants began in a controlled squat position with knees bent at 90 degrees and arms on hips. They held this position for 3 s before powering upwards in a maximal jump without any preparatory downward movement. The CMJ started from an upright standing position with feet shoulder-width apart and hands on hips. Participants then performed a controlled downward squat motion followed immediately by a forceful jump, maximising its vertical height. A standardised warm-up routine was performed before the fitness tests.

Finally, maximal aerobic capacity was evaluated using a progressive treadmill test (Ergofit Trac Alpin 4000, Pirmasens, Germany) with a gas analyser (Geratherm Respiratory GMBH, Ergostik, Ref 40.400, Corp, Bad Kissingen, Germany). Participants started by walking/running at a comfortable pace of 7 km/h on the treadmill with a 1% incline. The difficulty gradually increased with the speed rising by 1 km/h every minute until participants reached exhaustion. A 15 min warm-up at 6 km/h was performed before the main test.

2.7. Mn and Mo Determination

The methods used to extract Mn and Mo from various blood components followed established protocols similar to those employed in other research studies [21,30,31]. The method was developed entirely by the research support service of the University of Extremadura using inductively coupled plasma mass spectrometry (ICP-MS) (7900; Agilent Tech., Santa Clara, CA, USA). This method is recognised for its reliability, as demonstrated by calibration checks using the element indium. These checks consistently showed a strong correlation (greater than 0.985) and minimal variation (coefficients of variation less than 5%), signifying the accuracy and precision of the ICP-MS technique.

For plasma and urinary samples, the reagents used were 69% nitric acid (TraceSELECT™, Fluka™, Madrid, Spain) and ultrapure water obtained from a Milli-Q system (Millipore^®, Burlington, MA, USA). A rhodium dilution of 400 µgL⁻¹ was used as the internal standard and continuously fed into the apparatus with the aid of the three-channel peristaltic pump. From the 0.20 mL of samples, a volume of 5 mL was made up with a 1% nitric acid solution prepared from a commercial one of 69% (TraceSELECT™, Fluka™, Madrid, Spain). The equipment was calibrated with several standards prepared from commercial multi-elemental dilutions of certified standards.

For erythrocyte and platelet samples, the reagents used in method development and sample analysis were nitric acid 69%, hydrogen peroxide (TraceSELECT™, Fluka™, Madrid, Spain), and ultrapure water obtained from a Milli-Q system manufactured by Millipore (USA). A 400 µgL⁻¹ solution of yttrium and rhodium was used as the internal standard.

Samples were weighed on precision scales and transferred to glass tubes for microwave digestion, and 3.5 mL of a 3:1 mixture of 69% nitric acid (TraceSELECT™, Fluka™, Madrid, Spain) and hydrogen peroxide (TraceSELECT™, Fluka™, Madrid, Spain) was added. The samples were digested in a Milestone Ultrawave microwave, and once digested, were diluted to 25 mL with Milli-Q water. The detection and quantification limits of Mn and Mo in the different matrices throughout the investigation are shown in

Table 1. Limits of detection and limits of quantification.

Matrix	L. D. Mn 1 (µg/L)	L. Q. Mn 2 (µg/L)	L. D. Mo 3 (µg/L)	L. Q. Mo 4 (µg/L)
Plasma	0.001	0.01	0.018	0.18
Urine	0.005	0.05	0.002	0.02
Erythrocytes	0.002	0.02	0.004	0.04
Platelets	0.006	0.06	0.0016	0.02

¹ Limits of detection of manganese; ² limits of quantification of manganese; ³ limits of detection of molybdenum; ⁴ limits of quantification of molybdenum.

.

2.8. Statistical Analysis

IBM SPSS Statistics 25.0 software (IBM Corp., Armonk, NY, USA) was used to analyse the collected data. The results are presented as average values with an indication of variability (standard deviation). The normality of the variables was analysed using the Shapiro–Wilk test.

A two-way ANOVA was used to analyse most other variables. This test considers the effects of two factors: sex (male or female) and the time of measurement. The Bonferroni post hoc test was applied for the measured effect variable. Effect size was calculated using partial eta-squared (ƞ²), where 0.01–0.06 was a small effect size, 0.06–0.14 was a moderate effect size and >0.14 was a large effect size [32]. Differences of p < 0.05 and p < 0.01 were considered statistically significant and highly significant, respectively.

3. Results

Table 2. Results obtained from the physical condition tests.

		Men’s Soccer Players	Women’s Soccer Players	Sex Effect	Time Effect	Sex × Time
SJ (cm)	1st assessment	50.52 ± 6.48	35.65 ± 5.82	<0.001	0.515	0.602
	2nd assessment	49.73 ± 4.21	37.08 ± 5.14
	3rd assessment	50.90 ± 6.2	38.00 ± 5.49
CMJ (cm)	1st assessment	56.94 ± 6.39	40.21 ± 7.46	<0.001	0.571	0.717
	2nd assessment	55.34 ± 4.72	39.70 ± 4.18
	3rd assessment	56.05 ± 6.39	41.45 ± 5.80
Speed (km/h)	1st assessment	19.17 ± 1.72	15.73 ± 1.16	<0.001	0.289	0.315
	2nd assessment	19.22 ± 1.44	15.20 ± 1.10
	3rd assessment	19.15 ± 1.98	14.91 ± 1.37
VCO2max (L/min)	1st assessment	4.05 ± 0.36	2.68 ± 0.44	<0.001	0.377	0.710
	2nd assessment	3.85 ± 0.80	2.4 ± 0.31
	3rd assessment	53.30 ± 5.11	41.06 ± 4.51
VO2max (mL/min/kg)	1st assessment	52.21 ± 2.91	39.72 ± 6.22	<0.001	0.032	0.268
	2nd assessment	54.79 ± 3.70 *	42.32 ± 4.19 *
	3rd assessment	53.30 ± 5.11	41.06 ± 4.51

* p < 0.05 differences between 1st and 2nd assessment; SJ: squat jump; CMJ: countermovement jump; HR: heart rate; VO_2max: maximal oxygen consumption; VCO2max: maximal carbon dioxide production.

shows the results obtained in the physical condition tests. There were highly significant differences between sexes in all the parameters analysed (p ≤ 0.01). Maximal oxygen consumption (VO_2max) was higher in the second assessment in both groups (p < 0.05).

Table 3. Mean intake of the three days of evaluation of macronutrients, Mn, and Mo throughout the study.

		Men’s Soccer Players	Women’s Soccer Players	Sex Effect	Time Effect	Sex × Time
Energy (Kcal)	1st assessment	1796.0 ± 420.0	1578.1 ± 316.2	0.038	0.497	0.317
	2nd assessment	1932.2 ± 312.5	1681.5 ± 427.3
	3rd assessment	1882.7 ± 358.6	1697.3 ± 386.1
Proteins (g)	1st assessment	106.1 ± 25.5	90.4 ± 21.6	0.047	0.469	0.218
	2nd assessment	115.5 ± 23.4	96.2 ± 18.3
	3rd assessment	108.9 ± 24.8	92.6 ± 20.4
Proteins (g/Kg/day)	1st assessment	1.50 ± 0.39	1.27 ± 0.31	0.033	0.211	0.345
	2nd assessment	1.63 ± 0.34	1.36 ± 0.32
	3rd assessment	1.55 ± 0.42	1.30 ± 0.30
Lipids (g)	1st assessment	54.8 ± 19.1	48.3 ± 12.3	0.116	0.241	0.471
	2nd assessment	64.1 ± 15.4	55.6 ± 15.3
	3rd assessment	58.6 ± 17.4	60.3 ± 20.6
Lipids (g/Kg/day)	1st assessment	0.77 ± 0.29	0.71 ± 0.18	0.061	0.305	0.561
	2nd assessment	0.92 ± 0.21	0.76 ± 0.22
	3rd assessment	0.83 ± 0.24	0.81 ± 0.25
Carbohydrates (g)	1st assessment	231.0 ± 69.1	206.1 ± 81.3	0.471	0.856	0.683
	2nd assessment	235.8 ± 60.3	241.5 ± 56.1
	3rd assessment	242.0 ± 57.0	235.8 ± 61.7
Carbohydrates (g/Kg/day)	1st assessment	3.28 ± 1.09	3.03 ± 0.99	0.236	0.471	0.438
	2nd assessment	3.35 ± 0.95	3.48 ± 1.01
	3rd assessment	3.45 ± 0.97	3.30 ± 1.11
Mn (mg)	1st assessment	2.5 ± 1.1	1.9 ± 0.6	0.258	0.732	0.487
	2nd assessment	2.7 ± 1.3	2.2 ± 0.5
	3rd assessment	2.7 ± 1.6	2.1 ± 0.4
Mo (µg)	1st assessment	240.9 ± 99.1	201.5 ± 67.2	0.175	0.905	0.618
	2nd assessment	246.8 ± 76.0	214.7 ± 53.1
	3rd assessment	253.8 ± 97.7	241.7 ± 87.5

Mn: manganese; Mo: molybdenum.

shows the results obtained in the intake of macronutrients Mn and Mo throughout the season in both teams. Significant differences were observed between sexes in energy and protein intake, both being higher in the men’s soccer players (p ≤ 0.05).

Table 4. Erythrocyte and platelet values according to sex throughout the study.

		Men’s Soccer Players	Women’s Soccer Players	Sex Effect	Time Effect	Sex × Time
Erythrocytes (millions)	1st assessment	4.92 ± 0.36	4.37 ± 0.22	<0.001	0.031	0.063
	2nd assessment	4.83 ± 0.32 **	4.19 ± 0.27 **
	3rd assessment	4.99 ± 0.29 ++	4.35 ± 0.27 ++
Platelets (thousands)	1st assessment	204.50 ± 57.65	196.00 ± 38.01	0.274	0.542	0.222
	2nd assessment	196.60 ± 39.79	219.08 ± 34.19
	3rd assessment	195.13 ± 37.82	204.39 ± 31.52

** p ≤ 0.01 differences between 1st and 2nd assessment; ++ p ≤ 0.01 differences between 1st and 3rd assessment.

shows the values obtained for erythrocytes and platelets. There were differences between sexes in erythrocytes, being higher in the men’s soccer players (p < 0.01). The concentration of erythrocytes decreased in the second evaluation in both groups (p < 0.05).

Table 5. Mn concentrations in the compartments evaluated throughout the study in the two study groups.

		Men’s Soccer Players	Women’s Soccer Players	Sex Effect	Time Effect	Sex × Time
Mn Plasma (µg/L)	1st assessment	3.05 ± 1.48	0.94 ± 0.43	<0.001 #	0.182 $	0.061
	2nd assessment	2.00 ± 0.30	1.32 ± 0.28
	3rd assessment	1.69 ± 0.55	2.05 ± 0.49
Mn Urine (µg/L)	1st assessment	0.228 ± 0.196	0.088 ± 0.185	0.005 #	<0.001 #	0.055 $
	2nd assessment	0.565 ± 0.102 **	0.381 ± 0.172 **
	3rd assessment	0.421 ± 0.197 ++	0.433 ± 0.261 ++
Mn Erythrocyte absolute (µg/L)	1st assessment	40.40 ± 12.60	65.45 ± 24.78	0.002 #	0.007 #	0.150
	2nd assessment	50.95 ± 7.52 ^^	54.90 ± 10.14 ^^
	3rd assessment	44.56 ± 5.22 ++	47.01 ± 5.76 ++
Mn Erythrocyte relative (pg/cell-6)	1st assessment	8.60 ± 2.57	15.46 ± 5.39	<0.001 #	0.019 $	0.079 $
	2nd assessment	10.99 ± 1.59	13.15 ± 2.21
	3rd assessment	9.27 ± 1.16 ++	12.02 ± 1.34 ++
Mn platelets absolute (µg/L)	1st assessment	10.01 ± 4.26	15.25 ± 13.98	0.245	0.037 #	0.079 #
	2nd assessment	12.37 ± 3.37	13.20 ± 3.47
	3rd assessment	13.21 ± 2.48 ++	12.41 ± 4.76 ++
Mn platelets relative (pg/cell-3)	1st assessment	0.071 ± 0.025	0.079 ± 0.059	0.256	<0.001 #	0.132
	2nd assessment	0.065 ± 0.022 **	0.059 ± 0.012 **
	3rd assessment	0.071 ± 0.031	0.54 ± 0.022

#: large effect size (>0.14); $: moderate effect size (0.6–0.14); ** p ≤ 0.01 differences between 1st and 2nd assessment; ++ p ≤ 0.01 differences between 1st and 3rd assessment; ^^ p ≤ 0.01 differences between 2nd and 3rd assessment; Mn: manganese; Mo: molybdenum.

shows the data obtained on Mn concentrations in the extracellular and intracellular compartments. Men’s soccer players showed higher plasma and urinary concentrations. There were also differences across assessments in both compartments (p < 0.05).

In relation to erythrocyte Mn concentrations, there were differences between sexes and throughout the assessment in erythrocyte concentrations (p ≤ 0.05). Concentrations were higher in the women’s soccer players. Regarding differences throughout the investigation, specific differences were observed between assessment 1 and 3 and assessment 2 and 3 (p ≤ 0.05).

Finally, with respect to intraplatelet Mn concentrations, differences were observed throughout the season (p ≤ 0.05). Differences were found between assessments 1 and 3 expressed in absolute values, and between assessments 1 and 2 expressed in relative values (p ≤ 0.01).

Table 6. Mo concentrations in the compartments evaluated throughout the study in the two study groups.

		Men’s Soccer Players	Women’s Soccer Players	Sex Effect	Time Effect	Sex × Time
Mo Plasma (µg/L)	1st assessment	2.07 ± 0.59	1.44 ± 0.71	<0.001 #	<0.001 #	0.216
	2nd assessment	2.67 ± 0.75 **	1.93 ± 0.45 **
	3rd assessment	2.59 ± 0.79 ++	2.39 ± 0.49 ++
Mo Urine (µg/L)	1st assessment	53.98 ± 40.77	57.55 ± 46.16	0.066 #	<0.001 #	0.057 $
	2nd assessment	67.16 ± 37.14 ^^	34.33 ± 24.88 ^^
	3rd assessment	24.71 ± 16.63 ++	19.23 ± 13.80 ++
Mo Erythrocyte absolute (µg/L)	1st assessment	19.87 ± 6.15	30.97 ± 33.61	0.730	0.003 #	0.088 $
	2nd assessment	42.45 ± 37.61 **	44.37 ± 48.36 **
	3rd assessment	26.73 ± 20.11 ++	22.85 ± 9.15 ++
Mo Erythrocyte relative (pg/cell-6)	1st assessment	2.66 ± 1.01	3.50 ± 5.38	0.574	0.002 #	0.121
	2nd assessment	8.77 ± 7.99 **	10.26 ± 10.59 **
	3rd assessment	5.60 ± 3.98 ^^	4.58 ± 1.68 ^^
Mo platelets absolute (µg/L)	1st assessment	7.51 ± 2.51	8.71 ± 4.18	0.346	0.041 #	0.217
	2nd assessment	6.94 ± 2.98 ^^	8.15 ± 5.32 ^^
	3rd assessment	3.88 ± 1.53 ++	4.51 ± 1.42 ++
Mo platelets relative (pg/cell-3)	1st assessment	0.040 ± 0.014	0.042 ± 0.017	0.991	<0.001 #	0.996
	2nd assessment	0.032 ± 0.015 ^^	0.037 ± 0.027 ^^
	3rd assessment	0.024 ± 0.021 ++	0.028 ± 0.005 ++

#: large effect size (>0.14); $: moderate effect size (0.6–0.14); ** p ≤ 0.01 differences between 1st and 2nd assessment; ++ p ≤ 0.01 differences between 1st and 3rd assessment; ^^ p ≤ 0.01 differences between 2nd and 3rd assessment; Mo: molybdenum.

shows the data on Mo concentrations in the extracellular and intracellular matrices. Men’s soccer players showed higher plasma and urinary concentrations (p < 0.05). Urinary and plasma Mo concentrations decreased and increased, respectively, at the end of the study (p < 0.05).

For intracellular concentrations in both platelets and erythrocytes, differences were observed across assessments (p ≤ 0.05). Differences in erythrocyte Mo concentrations were found between assessments 1 and 2, 1 and 3, as well as assessments 2 and 3 (p ≤ 0.01). On the other hand, with respect to intraplatelet Mo concentrations, both in absolute and relative values, differences were observed between assessments 1 and 3 and assessments 2 and 3 (p ≤ 0.01).

4. Discussion

The objectives of the present work were: (a) to analyse the changes in Mn and Mo concentrations in plasma, urine, erythrocytes, and platelets during a sports season in soccer players and (b) to analyse the differences between sexes. To our knowledge, this is one of the first studies to analyse Mn and Mo concentrations during a season in different biological matrices. Information regarding these minerals in athletes is scarce, which highlights the novelty of the present study. For a better understanding of this section, the results obtained on Mn and Mo concentrations will be discussed separately.

Mn and Mo absorption could be influenced by Fe concentrations. Gastrointestinal Mn absorption involves the main intestinal Fe transporters such as DMT1. The activity of this transporter increases when Fe stores are low, which would explain the increased Mn absorption under Fe deficiency conditions [33]. On the other hand, the functioning of most of the molybdoenzymes depends on Fe metabolism to provide Fe sulphide groups and heme groups. The main interrelationship is the role that XO could play in the release of Fe from ferritin [34].

Mn reference intakes established by sex (mg/day) usually vary between 3.4 mg/day for men and 2.7 mg/day for women [35]. Mn concentrations in athletes have been previously analysed in serum, urine [18,19], and erythrocytes [36]. However, the literature regarding platelets is scarce. Mn concentrations in this research were within the range reported in other studies [37,38].

When differences were analysed along the assessment in the extracellular compartments, no differences in plasma concentrations were observed. However, there was an increase in urinary Mn excretion in both groups. In relation to urinary Mn values, increases in excretion were previously reported in athletes after a six-month training period [18]. Likewise, in the previous study, they observed a decrease in serum Mn concentrations after six months of training. However, when the acute effect of physical exercise was evaluated, there was a decrease in Mn excretion [18,19].

Both the level of training and the type of sport could affect Mo concentrations.

Lower erythrocyte concentrations have been observed in athletes with high levels of training. This could be due to the absence of Mn-SOD inside the erythrocyte because there are no mitochondria in this cell [39]. On the other hand, depending on the type of sport, higher serum concentrations have been observed in aerobic athletes. The higher Mn concentrations in athletes could be related to a higher organic utilisation of this mineral in mitochondrial Mn-SOD production processes, among others. The higher basal blood Mn concentrations in aerobic athletes compared to athletes of other sports could be related to probably lower Fe levels, a fact that is common in long-distance athletes, as a consequence of the impact of body Fe levels on Mn metabolism [20].

The gradual low plasma concentration observed in the present investigation could be caused by an increase in Fe absorption [22,36], which would be related to increased Mn excretion, revealing a possible renal adaptation with training. Another reason for decreased plasma Mn concentrations could be an increase in Mn-SOD activity, which has been shown to increase at the end of the season [40]. As mentioned above, free radicals are produced as a result of oxidative metabolism. Increased free radicals are associated with increased lipid peroxidation and accumulation of metabolites that can lead to injury and cell death. Among other enzymes, Mn-SOD controls free radical damage, and it is known that one of the adaptations to physical training is an increase in its activity [41].

As for intracellular concentrations, there were different changes according to sex. In the men, a gradual increase of Mn concentrations in platelets and erythrocytes was observed. However, in the women, decreases in Mn concentrations were observed in both intracellular compartments. No studies have been found that evaluate the evolution over a training season/period of erythrocyte and platelet Mn concentrations. Previously, it has been reported that sedentary subjects showed higher erythrocyte Mn concentrations compared with active or very physically active populations [36]. It should be noted that Mn-SOD would exist in very small proportions inside erythrocytes, since there are no mitochondria in this type of cell. Therefore, the evolution in erythrocyte Mn concentrations could be due to the different Mn intake between sexes [36]. Regarding platelet Mn concentrations, there are no reports in the scientific literature on Mn concentrations in this compartment throughout the season in athletes. Perhaps, as occurs with erythrocytes, sex differences in intake could explain the trend of Mn in this biological matrix.

Regarding sex differences, higher Mn concentrations have been observed in plasma and urine in men compared to women. Despite absorbing less Mn, absorbed Mn had a longer half-life in the men compared to the women [42]. However, in a population of 1417 U.S. subjects, it was observed that the women excreted more Mn relative to urinary creatinine values [43].

As mentioned above, Fe deficiency influences Mn metabolism by increasing absorption. Since the main route of Mn elimination is bile [42], these data may show that hepatic secretion of Mn into bile is more active in women than in men. The influence of sex on whole-organism Mn turnover warrants further investigation of whether sex influences hepatic metabolism of Mn and its excretion in bile. It has been shown that the hepatocyte moves Mn into the bile canaliculus against a concentration gradient. Therefore, we believe that the lower extracellular Mn concentration may be due to an increased elimination of Mn through bile in women’s soccer players. Mn is essential for reproductive function, as it is a cofactor for the enzymes mevalonate kinase, geranyl pyrophosphate synthetase, and farnesyl pyrophosphate synthetase, which are necessary for the synthesis of cholesterol, which is important for the synthesis of steroid hormones, such as estrogens [44].

Regarding erythrocyte concentrations, women showed higher erythrocyte concentrations compared to men, both in relative and absolute values. The data are in agreement with those previously reported in a study with more than 7700 Americans [45], Canadians [46], and Koreans [47]. According to the authors, this could be due to a lower blood ferritin concentration in women [47]. Higher blood Mn levels were also associated with lower whole blood Fe levels in persons older than 12 years [45]. This suggests that there may be sex-related metabolic differences in the homeostatic mechanisms regulating blood Mn levels. A study on dietary Mn absorption showed that, when consuming a diet adequate in Mn, women can absorb significantly more Mn than men [42].

Mo reference intakes established by sex (µg/day) usually vary between 109 µg/day for men and 76 µ/day for women [35]. The Mo concentrations analysed in different compartments were within the ranges reported in previous investigations [36,38]. Mo is of relatively little interest to physically active individuals [48]. As discussed above, Mo is a transition element that readily changes its oxidation state, and thus can act as an electron transfer agent in oxidation–reduction reactions [48].

Different markers have been used to assess Mo status. Plasma and serum Mo concentrations are low in humans and therefore complex to assess. As a consequence, there are few studies on Mo concentrations in these biological compartments [49].

Differences have been observed throughout the measurements in plasma Mo concentrations, increasing with respect to the first assessment. Previous authors reported that serum Mo concentrations increased after performing 2 km on an ergometer [49]. They also reported increased concentrations after a marathon [50]. On the other hand, other authors did not observe significant changes in serum Mo concentrations after an aerobic training period of 6 months [18]. However, they observed that athletes had higher serum concentrations compared to the control group. When analysing basal states, they reported that athletes showed higher serum concentrations compared to the control group, with anaerobic athletes having the highest concentrations within the athlete group [18,49,50]. With respect to urinary concentrations, no significant changes were observed after a six-month training period [18]. Similarly, when the acute effect of a maximal incremental test was analysed, no differences were observed in Mo excretion [22].

The increase of Mo concentrations in plasma and urine throughout the assessments in the study participants could facilitate the formation of uric acid, avoiding the damage of free radicals (superoxide anions) generated by XO in the ischaemia-reperfusion processes generated during high-intensity physical exercise [13].

With respect to the changes observed throughout the evaluations in the intracellular Mo concentrations, increases were observed in the middle of the season and a decrease at the end of the season in the erythrocyte concentrations. On the other hand, progressive decreases in intraplatelet concentrations were shown. At basal state, medium- and high-level athletes showed lower intracellular Mo concentrations compared to the control group [22,36]. No information has been found regarding erythrocyte and platelet concentrations of Mo in athletes.

Regarding sex differences, the present study showed that the men’s soccer players had higher plasma Mo concentrations compared to the women’s soccer players. It is known that uric acid concentrations appear to be higher in active men [51] and in men athletes [52,53]. This could be due to differences in plasma Mo concentrations, since, as discussed above, Mo facilitates the formation of uric acid to prevent free radical damage. In addition, women seem to be less susceptible to oxidative stress, specifically premenopausal women, due to the antioxidant role of estrogens, and therefore uric acid levels could be lower [54].

Study Limitations

The present study is not free of limitations: (a) Plasma volume was not evaluated. During physical exercise, body water can be lost through sweating, which can induce dehydration, blood haemoconcentration, and a decrease in urine water, which could influence the results obtained; (b) an absence of complementary data of the different MTEs (enzymes); (c) the small sample size; (d) technical measurement error was not analysed; and (e) we evaluated a nutritional level and not the athletes’ eating habits.

5. Conclusions

Plasma and urinary Mn concentrations were higher in the men’s soccer players, whereas erythrocyte Mn concentrations were higher in the women’s soccer players. Increases in urinary, erythrocyte, and platelet concentrations occurred in the men throughout the sports season, whereas in the women’s soccer players, a decrease in erythrocyte and platelet concentrations was observed with respect to the initial values.

Plasma Mo concentrations were higher in the men’s soccer players. Throughout the sports season there were increases in plasma and erythrocyte concentrations, as well as decreases in urinary and platelet concentrations of Mo with respect to the initial values in both sexes.