Measuring trace element concentrations in human hair serves as an important analytical method for screening potential deficiencies, excesses, or biochemical imbalances of these microelements in the body. In this research, using an inductively coupled plasma–mass spectrometry (ICP-MS) analyzer, the authors aimed to identify toxic trace elements (Al, Pb, Hg) and quantify key mineral elements (Ca, Mg, Cu, Zn) in healthy individuals with good nutritional status. The study included a sample of 75 adult women aged 30–35 years from various regions across the country. Hair samples weighing 100 mg and measuring 3 cm from the scalp base were collected for trace element analysis and evaluation. Results showed that 12 participants (16%) exhibited elevated intracellular Mg levels (1.2 mmol/L), increased Ca levels (0.72 mmol/L), yet decreased mean Ca/Mg ratios (0.58). Conversely, 6 participants (8%) demonstrated low mean values of Mg (0.004 mmol/L) and Ca (0.04 mmol/L) but had a high Ca/Mg ratio. At the time of analysis, none of the subjects displayed signs of acute or severe heavy metal intoxication. The hair trace element content reflected the environmental and lifestyle factors of the individuals from different regions of the country.
Mineral analysis of hair traces serves as a valuable tool for evaluating overall nutrient status and health [1–3]. Assessing trace elements in hair can aid in diagnosing conditions such as human poisoning, cancer, and malnutrition [4, 5], benefiting from the ease of sample collection and long-term storage.
Hair mineral analysis involves measuring the concentrations of both nutritional and toxic mineral elements present in hair tissue. A key aspect of this test is determining mineral levels stored within hair cells and the interstitial spaces over a 2–3 month period, which helps infer mineral status in other tissues. However, it does not directly reflect mineral content in different body tissues [6]. While blood tests measure elements that are absorbed and transiently circulate before excretion or storage, hair analysis records a lasting exposure profile, with element concentrations in hair typically 10–15 times higher than those found in blood or urine.
Hair analysis is also a significant method for biological monitoring of environmental pollution [7]. It can be used to encourage individuals to maintain a healthy lifestyle in their surroundings, in line with the United Nations Global Environment Monitoring System (GEMS) [8].
Numerous studies in the literature have described the quantification of inorganic components in human hair, revealing correlations between mineral composition and common diseases. For example, blood pressure levels have been assessed alongside concentrations of Fe, Ca, Mg, Zn, Cu, Na, and K [9]; diabetic patients exhibited notably lower manganese and zinc levels [10]; and lung cancer patients showed elevated cadmium concentrations in scalp hair compared to controls, with smokers presenting particularly high cadmium levels relative to non-smokers [11].
The morphological condition of hair from individuals with diabetes, hypertension, cholelithiasis, and kidney stones has been examined using scanning electron microscopy [12].
The inductively coupled plasma–mass spectrometry (ICP-MS) technique, applied for trace element analysis in bulk products and human tissues, is effectively utilized to determine mineral concentrations in human hair for purposes including nutritional assessment, toxicology, and pharmaceutical drug trials [13–15].
In this research, using an inductively coupled plasma–mass spectrometry (ICP-MS) analyzer, the authors aimed to identify toxic trace elements (Al, Pb, Hg) and quantify key mineral elements (Ca, Mg, Cu, Zn) in healthy individuals with good nutritional status.
The primary aim of this study was to assess the intracellular levels of key mineral nutritive elements (Ca, Mg, Cu, Zn) and trace elements (Al, Pb, Hg) in human hair using an inductively coupled plasma–mass spectrometry (ICP-MS) instrument, while examining the relationship of trace element status with potential human health risks. Analysis of metal content, including the main mineral nutritive elements (Ca, Mg, Cu, Zn) and trace elements (Al, Pb, Hg), was conducted using a conventional ICP-MS (PerkinElmer 900). Residual digested hair samples weighing 100 mg were analyzed, and results were expressed with the equivalence: 1 part per million (ppm) = 1000 parts per billion (ppb) = 1 mg/L.
The study involved 75 adult females, aged 30 to 55 years, from various regions of the country. Hair samples of 100 mg, cut from the first 3 cm nearest the scalp, were placed in special ICP-MS cuvettes to provide information about nutrient status over the past 6 to 8 weeks.
ICP-MS calibration was performed using 10 milliliters of 60% concentrated nitric acid (HNO3) diluted at ratios of 1:10, 1:100, and 1:1000 under a pressure of 35 atm (315 psi). The sample blank solution was prepared as 200 milliliters at 2% concentration of the 60% HNO3 stock solution (calculated as 6.6 ml HNO3 plus 193.4 ml water).
Hair samples of 100 mg were collected by cutting the closest 3 cm segment to the scalp. Samples were mineralized with 10 ml of 60% concentrated HNO3 for 15 minutes in open-lip cuvettes, followed by a 1:1 volume dilution. Digestion continued in the ICP-MS instrument using micro-sonication at 1600 W power, heated for 15 minutes at a pressure of 800 psi. The final results were interpreted following chromatographic analysis using ICP-MS.
The findings, presented in Tables 1–3, are comparable to those reported in other international medical literature. All participants were registered and monitored for intracellular mineral element concentrations.
Out of the total participants, 55 (73%) exhibited normal intracellular levels: Mg (normal range = 4.1–10.5 ppb, equivalent to 0.03–0.09 mmol/L, mean = 0.06 mmol/L; SD = 0.2 mmol/L; p = 0.02); Ca (normal range = 20–40 ppb, equivalent to 0.1–0.2 mmol/L, mean = 0.15 mmol/L; SD = 2.5; p = 0.05); and a normal Ca/Mg ratio (range 4.5–7.5, mean = 6.5) as shown in Table 1.
Table 1. Database of ICP-MS; Perkin Elmer 900 [16].
No patient | Concentrations (mean value) | Reports mean value | |||
No. Cr | Ca (ppb) | Ca (mmol/L) | Mg (ppb) | Mg (mmol/L) | Ca/Mg (ppb) |
55 | 397.50 | 0.15 | 81.48 | 0.06 | 4.87 ppb |
12 | 558.45 | 0.29 | 335.8 | 0.4 | 1.66 ppb |
8 | 149.50 | 0.07 | 200.1 | 0.02 | 0.74 ppb |
Reference | 22-97 ppm male 18-47 ppm female |
| 2-11 ppm 2-18 ppm |
| 6.77 ppb 6.80 ppb |
Twelve patients (16%) demonstrated significantly elevated intracellular Mg levels, with a mean of 1.2 mmol/L, alongside high Ca concentrations averaging 0.72 mmol/L, but exhibited a low Ca/Mg ratio, averaging 0.58. Conversely, eight patients (8%) showed reduced intracellular Mg (mean = 0.004 mmol/L) and Ca levels (0.04 mmol/L), but a markedly high Ca/Mg ratio of 10.
Notably, nine patients had normal Ca levels measured by mass spectrometry (0.18 mmol/L), extremely low Mg levels (0.006 mmol/L), and an exceptionally high Ca/Mg ratio of 30. For these particular cases, blood tests assessing Ca and Mg were also performed: serum total Ca measured 10.7 mg/dL (2.30 mmol/L) within the normal range (9.1–10.8 mg/dL or 2.19–2.54 mmol/L), and ionized Ca²⁺ was 1.41 mmol/L (normal range 1.02–1.42 mmol/L). Blood Mg was recorded at 2.53 mg/dL (1.04 mmol/L), also within normal limits (1.6–2.5 mg/dL or 0.73–1.06 mmol/L). Although blood Ca and Mg values were normal, the elevated intracellular Ca/Mg ratios suggest alterations in the physiopathological regulation of Ca and Mg ion dynamics, which are critical for cellular function.
The analysis of Cu and Zn levels in hair samples from selected patients, essential trace elements for assessing nutritional and health status, is summarized in Table 2. Both Zn and Cu were found at elevated concentrations; however, the Zn/Cu ratio remained within the normal range, as determined by ICP-MS chromatographic analysis. Patients with increased Zn and Cu levels frequently reported muscle spasms, especially nocturnal cramps, often occurring under conditions of intense physical stress. These findings align with reference ranges from other licensed clinical laboratories: for example, Zn typically ranges from 10 to 21 ppm; Cu ranges from 0.9 to 3.9 ppm; and the acceptable Zn/Cu ratio falls between 4 and 12. A sample measurement for a 47-year-old female showed Zn at 16 ppm, Cu at 0.9 ppm, and a Zn/Cu ratio of 17.78 ppm (Table 2).
Table 2. Level of intracellular concentration of trace elements
No patient | Concentrations (mean value) | Reports mean value | |||
| Zn (ppm) | Zn (µg/dL) | Cu (ppm) | Cu (µg/dL) | Zn/Cu (µg/dL) |
8 | 13.52 | 42.3 | 5.83 | 1.83 | 23.6 |
7 | 10.13 | 32.1 | 4.78 | 1.5 | 21.4 |
10 | 10.48 | 3.2 | 13.4 | 4.2 | 2.57 |
15 | 28.71 | 8.7 | 11.69 | 3.67 | 7.8 |
17 | 8.47 | 19.1 | 18.96 | 5.95 | 13.8 |
18 | 3.21 | 10.7 | 3.46 | 1.1 | 32 |
Reference range | 10-21 |
| 0.9-3.9 |
| 4-12 |
Table 3 displays selected data illustrating elevated concentrations of metals with toxic potential found in hair samples.
Table 3. Intracellular concentration of toxic elements (N = normal values; H = high values).
Hg | Pb | Al | |||
C (µg/dL) | Bounding | C (µg/dL) | Bounding | C (µg/dL) | Bounding |
0.84 | N | 0 | N | 0.11 | N |
0.63 | N | 0.17 | N | 0.09 | L |
3.5 | H | 0 | N | 0.2 | N |
0 | N | 0.03 | N | 0.3 | H |
0 | N | 0 | N | 0.23 | H |
0.18 | N | 0 | N | 0.2 | N |
0.18 | N | 0.01 | N | 0.3 | H |
0.21 | N | 0 | N | 0.4 | H |
0.5 | N | 0.02 | N | 0.8 | H |
0.12 | N | 0 | N | 0.4 | H |
0.25 | N | 0 | N | 0.15 | N |
0.44 | N | 0 | N | 0.2 | N |
2.46 | H | 0 | N | 4.9 | H |
1.29 | H | 0 | N | 1.8 | H |
0.042 | N | 0 | N | 0.1 | L |
2.46 | H | 0 | N | 0.5 | H |
1.29 | H | 0 | N | 0.7 | H |
0.042 | N | 0 | N | 0.4 | H |
0.55 | N | 0.88 | H | 3.42 | H |
0.63 | N | 0 | N | 0.4 | H |
Based on the normal reference ranges for the method used—mass spectrometry (MS Agilent)—the values are as follows: Hg (normal range = 0.63–0.99 µg/dL), Pb (normal range = 0.3–0.45 µg/dL), and Al (normal range = 0.15–0.21 µg/dL). Among the 75 individuals analyzed, the distribution of values was: for Hg, 15 individuals had normal levels and 5 had elevated levels; for Pb, 19 individuals showed normal levels and 1 had a high level; and for Al, 4 had normal levels, 14 had elevated levels, and 2 showed low levels.
Calcium (Ca²+) and magnesium (Mg²+) are essential mineral elements involved in numerous enzymatic reactions within both intracellular and extracellular processes, including blood coagulation, aerobic cellular metabolism, and the coordinated function of various organ systems and endocrine glands necessary for maintaining overall health.
Only about 1% to 3% of total intracellular magnesium exists as the free ionized Mg²+ form, which is tightly regulated within a concentration range of 0.05 to 0.1 mmol/L. The findings in our study generally did not align with this range (except for one particular case, as indicated in point 2 of (Table 1)), revealing a magnesium deficiency in many samples, especially among those listed as point 4 in the table, where Mg levels were as low as 0.006 mmol/L. Total cellular magnesium concentrations vary between 0.55 and 2 mmol/dL depending on the tissue type, with the highest levels found in skeletal and cardiac muscle cells [17].
Magnesium is the second most abundant intracellular cation after potassium, playing a crucial role in regulating diverse cellular functions and enzymes, including signaling pathways, metabolic cycles, and ion channels.
Mg²+ ions are vital for maintaining the structural integrity of densely packed phosphate groups, which occur in various regions of the cytoplasm and nucleus. Mg²+ also stabilizes proteins, ribosomes, and nucleic acids, and serves as an essential trace element involved in energy metabolism.
Cell walls and biological membranes exhibit poly-anionic charges on their surfaces, and since different membranes preferentially bind specific ions, this has important implications for ion transport. To stabilize Ca²+ and Mg²+ in membranes, the phosphorylated and carboxylated lipid head groups form cross-links.
In pathological conditions such as malignancies, cancer cells utilize Mg²+ ions more extensively than normal cells, increasing magnesium uptake from normal tissue stores, including muscle and bone. Serum magnesium levels can rise due to Mg²+ release from malignant tissues in patients with cancer before starting cytostatic chemotherapy.
Copper (Cu) and zinc (Zn) play significant roles in maintaining proper physiological function throughout the body. For example, patients with epilepsy have been shown to have reduced mean hair concentrations of Zn and Cu [18]. Specifically, hair levels of Mg, Zn, and Cu (measured in ppm) were significantly lower in epilepsy patients compared to controls: Mg (111.33 ± 37.33 vs. 133.57 ± 22.91; P < 0.01), Zn (121.40 ± 45.40 vs. 176.96 ± 43.10; P < 0.001), and Cu (42.74 ± 20.36 vs. 60.22 ± 22.32; P < 0.05).
Zinc is a crucial trace element involved in supporting the immune system and neurogenesis. A zinc deficiency may negatively impact cognitive function, attention span, and motor skill development [19]. Hair zinc concentrations in students with lower IQ scores were found to be reduced (141.70 ± 88.56 µg/g) in comparison to those of students with average IQ levels (198 ± 90.90 µg/g), with a statistically significant difference (P = 0.01). In contrast, copper levels in the same groups showed less variation: learners with lower IQ had Cu levels of 11.90 ± 3.97 µg/g versus 13.23 ± 3.97 µg/g in the control group, with P = 0.18, indicating no significant difference [20].
A recent investigation compared the scalp hair concentrations of Zn, Ca, and Mg between children with growth retardation and a healthy control group. The results showed average Zn levels of 157 ± 25 µg/mL in the affected group compared to 1218 ± 42.29 µg/mL in controls (P = 0.001); Ca levels of 1168 ± 231 µg/mL in growth-delayed children versus 1417 ± 245 µg/mL in controls (P = 0.001); and Mg levels of 168 ± 50 µg/mL compared to 206 ± 40 µg/mL (P = 0.005). These results indicate that the concentrations of Zn, Ca, and Mg were significantly lower in the hair of children experiencing growth retardation compared to the control group (P < 0.05) [21].
Hair from children is frequently used as a biological marker to assess exposure to heavy metals. In one study, 40 hair samples from Libyan children were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) to determine concentrations of heavy metals (Cd, Pb, and Hg) in children diagnosed with autism and in neurotypical children. The analysis revealed higher levels of these toxic elements in the autistic group: Pb at 40.99 ± 32.02 µg/g versus 37.00 ± 14.61 µg/g; Cd at 0.82 ± 0.98 µg/g versus 0.42 ± 0.27 µg/g; and Hg at 7.84 ± 10.57 µg/g compared to 4.07 ± 2.54 µg/g in the control group [22].
Trace metal concentrations were analyzed in hair samples from children living in areas surrounding Athens, Greece [23]. The measured levels of heavy metals with toxic potential—attributable to environmental pollutants such as air and water—were reported as average values (μg/g) for Hg, ranging from 0.52 to 0.36, and for Pb, from 3.31 to 0.80. The heavy metal burden in hair reflects both environmental exposure and the absorbed dose, offering a reliable indication of exposure potential when correctly interpreted. Inhabitants of Krakow also underwent hair analysis, with the findings indicating potential environmental risks. The measured concentrations (mg kg⁻¹ of dry matter) were as follows: Zn—260.86 in females and 241.68 in males; Pb—2.46 in females and 3.04 in males, highlighting a hazard level akin to near-occupational exposure [24].
Approximately 60% of total magnesium ions are stored in bodily tissues, while the remaining 40% are involved in intermediary metabolic processes. Of these, nearly 70% exist in the ionized Mg²⁺ free form, whereas the other 30% are bound to citrate, phosphate, proteins (mainly albumin), or form other complexes. Serum magnesium concentrations are tightly regulated within a narrow physiological range (0.65–1.05 mmol/dL; 1.58–2.25 mg/dL), primarily through renal control in the ascending loop of Henle. The hydrated 3-D structure formed by Mg²⁺ ions in aqueous environments influences its transport dynamics.
Cancer remains a major diagnostic concern. Supporting this, Czerny et al. [25] examined the relationship between cancer occurrence and the status of trace elements in the body, assessed through hair mineral analysis. Using ICP-MS, metal concentrations were measured in Polish female cancer patients and grouped based on cancer type: malignancies with high glycolytic activity (D), alimentary tract cancers (HG), and hormone-dependent cancers (H). The data for these groups are detailed in Table 4.
Table 4. Content of tested elements in the hair of cancer patients
Metals | Concentration level (𝜇g/g ) | |||
Control (mean ± SD) | H (mean ± SD) | HG (mean ± SD) | D (mean ± SD) | |
Ca | 425.25 ± 80.93 | 800.12 ± 403.05∗∗∗ | 275.86 ± 102.89∗∗∗,+++ | 689.43 ± 282.36eee ∗∗∗ |
Hg | 27.16 ± 8.14 | 20.53 ± 14.06∗∗∗ | 9.58 ± 4.69∗∗∗,+++ | eee 15.77 ± 7.61∗∗∗ |
Cu | 13.15 ± 3.12 | 87 ±2.57∗∗∗,+++,† | 7.99 ± 4.22∗∗∗ | 11.45 ± 5.67∗∗∗ |
Zn | 141.23 ± 32.11 | 130.58 ± 39.01∗,+++ | 125.09 ± 47.28∗∗ | 74.55 ± 27.53∗∗∗,†††,eee |
Hg | 0.02 ± 0.02 | 0.04 ± 0.05∗∗∗ | 0.04 ± 0.06∗∗∗ | 0.06 ± 0.06∗∗∗ |
Pb | 0.85 ± 0.54 | 0.94 ± 0.53 | 1.18 ± 0.98∗ | 1.19 ± 0.91∗∗ |
Al | 1.10 ± 1.01 | 2.23 ± 2.5∗∗∗ | 3.37 ± 3.69∗∗∗ | 3.97 ± 4.65∗∗∗ |
Statistical difference versus control, 𝑃 < 0.001, 𝑃 < 0.01, 𝑃 < 0.05, respectively, +++, ++, +, statistical difference versus D group, 𝑃 < 0.001, 𝑃 < 0.01, 𝑃 < 0.05, respectively, †††, ††, †, statistical difference versus HG group, 𝑃 < 0.001, 𝑃 < 0.01, 𝑃 < 0.05, respectively, statistical difference versus H group, 𝑃 < 0.001, 𝑃 < 0.01, 𝑃 < 0.05, respectively, hormone-dependent cancer group (H).
The findings of this study highlight the value of using hair element analysis as an initial screening approach to detect various diseases in women across different age ranges. The composition of trace elements in hair tissue serves as a reflection of the subjects’ environmental exposure—such as geographic location, dietary practices, and the quality of consumed air and water—providing a window into systemic elemental imbalances across the body.
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This research involved volunteer participants who were enrolled in scientific courses conducted under the project TDM POSDRU/81/3.2/S/58819, within the framework of the program “Professional training system for medical staff in the field of new technologies in the health system, (molecular diagnosis).” The project was led by the National Institute of Research Victor Babes in Bucharest, Romania, and was officially sanctioned by the EU (www.tdm-dru.ro).
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