The Relationship between Changes in Serum Element Concentrations and Pathological Condition and Disease Status in Japanese Multiple Myeloma Patients: A Pilot Study and Literature Review.

Introduction

Multiple myeloma (MM) is a rare hematological cancer, accounting for approximately 1% of all cancers (Kazandjian 2016; Siegel et al., 2020). MM is characterized by the monoclonal proliferation of plasma cells with abnormal hematopoiesis, increased immunoglobulins called M-proteins, hypercalcemia, decreased renal function, anemia, and bone lesions. Bone lesions cause pain and decrease quality of life and have a negative impact on life expectancy (Zhang and Zhuang, 2022).

Trace elements in the body are well-balanced and play an important role in maintaining normal life functions (Wada, 2004; Nishito and Kambe, 2018; Mehri, 2020). Serum element concentrations have been shown to change in patients with leukemia and lymphoma (Stevens et al., 2011; Azarm et al., 2013; Shen et al., 2023). It has been reported that patients with acute leukemia have higher serum copper (Cu) concentrations and lower serum zinc (Zn) concentrations than healthy individuals, and the serum Cu concentration and Cu to Zn ratio (Cu/Zn ratio) are associated with survival in patients with acute leukemia (Demir et al., 2011; Li et al., 2023). Similarly, decreased serum Zn concentration and increased Cu concentration and Cu/Zn ratio have been reported in MM patients (Khadem-Ansari et al., 2018). It has also been reported that abnormal iron (Fe) metabolism is involved in anemia in MM (VanderWall et al., 2013), that serum Fe concentration affects the therapeutic efficacy of bortezomib (Campanella et al., 2013; Camiolo et al., 2020), and that there is a correlation between selenium (Se) levels and the risk of developing MM (Yuan et al., 2022). However, no studies have analyzed multiple element concentrations simultaneously in MM patients. Additionally, although MM is likely to relapse after remission, changes in serum element concentrations between initial diagnosis (newly diagnosed: ND) and relapse (relapsed and/or refractory: RR) in the same patient are not clear.

Therefore, as a pilot study, we aimed to investigate changes in serum element concentrations in Japanese MM patients by multi-element exhaustive analysis and to compare the levels in MM patients and those in non-MM volunteers. We also compared serum element concentrations of the same MM patients at diagnosis and relapse.

Materials and Methods

This study was approved by the Ethics Committee of Nagoya City University Graduate School of Medical Sciences (60-00-1118) and the Epidemiological Ethics Review Committee of the Aichi Prefectural Institute of Public Health (18E-07). Informed consent was obtained from all study participants in accordance with the Helsinki Declaration.

Study participants

The study was designed as a case-control study. We included 12 Japanese patients (5 men and 7 women) diagnosed with MM between 2008 and 2013 at the Nagoya City University Hospital. Patients who underwent blood transfusion prior to blood collection were excluded. Samples were collected from the same patients at diagnosis and at relapse. The control group included 9 volunteers (5 men and 4 women). Controls were recruited from non-MM volunteers of similar age and sex as the patients in the MM group, and those with hepatic or renal disease that could affect serum element concentrations were excluded. The administration of medication in the control group was confirmed through interviews with participants.

For each MM patient, information on age, sex, disease type, stage according to the Durie-Salmon Staging System (Durie and Salmon, 1975) and the International Staging System for MM (Greipp et al., 2005), common myeloma-related cytogenetic abnormalities, various laboratory values, dietary intake status, bone lesions based on computed tomography (CT) or magnetic resonance imaging (MRI), and medications was obtained from medical records. We also analyzed the correlation between serum element concentrations and laboratory values related to disease status and tumor volume. The control group did not include any patient treated at the study site. Hence, there were no laboratory data available for this study.

Measurement of serum element concentrations and their relation to MM

To examine the relationship between the development of MM and serum element concentrations, we compared the differences in serum concentrations of each element between the control and MM groups. We targeted 12 elements (chromium [Cr], manganese [Mn], Fe, Cu, Zn, arsenic [As], Se, rubidium [Rb], strontium [Sr], molybdenum [Mo], antimony [Sb], and barium [Ba]). Preliminary tests confirmed that these elements had good measurement accuracy.

In all study participants, blood was collected before breakfast at fasting status. In all MM patients, blood samples were taken during hospitalization, and they were put on a hospital diet of a certain caloric and nutritional composition. After blood collection, serum was separated, and stored frozen until element concentrations were determined. The Insepack ® Ⅱ-D (Kyokuto Pharmaceutical Industrial, Tokyo, Japan) blood collection tube was used for the control and MM groups. Since the specimens were stored, their element concentrations were compared in a preliminary experiment with those of specimens collected using BD Vacutainer ® (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) blood collection tubes, which are blood collection tubes for testing trace elements, to confirm that there was no elution of the target elements. The samples were diluted (1 + 99) in nitric acid and filtered through a 0.45-μm membrane filter if insoluble material was present. Element concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS; 7800, Agilent Technologies, Santa Clara, CA, USA). The reagents used were nitric acid 1.38 (Kanto Chemical, Tokyo, Japan) and ICP-MS mixing standard XSTC-622 (SPEX CertiPrep, Metuchen, NJ, USA).

Statistical analysis

For samples with element concentrations below the lower limit of quantification (0.20 μg/L), half the lower limit of quantification, 0.10 μg/L, was used for statistical processing. Steel’s method was used to compare controls and MM patients (at diagnosis and relapse), and the Wilcoxon signed-rank test was used to compare MM patients at diagnosis and at relapse (the ND and RR groups, respectively). Correlation coefficients were determined using Spearman’s correlation coefficient. Differences were considered significant at P < 0.05. Statistical analyses were performed using the SPSS software package (version 17.0, SPSS Inc., Chicago, IL, USA) and EZR (version 1.54, Saitama Medical Center, Jichi Medical University, Saitama, Japan) (Kanda, 2013).

Results

Patient information

The baseline characteristics of the study participants are presented in Table 1 . The mean age of the control group was 66.6 ± 14.7 years, and the mean ages of the MM patients at diagnosis and relapse were 64.0 ± 10.3 years and 65.8 ± 10.1 years, respectively. Based on the radiologist’s evaluation by CT or MRI, bone lesions were found in 10 of 12 MM patients at diagnosis. Two patients had no obvious bone lesions at diagnosis or relapse. There were no grade 3 cases of decreased dietary intake (limitation of self-care activities of daily living) (National Cancer Institute, 2017).

Table 2 shows the median and range of the laboratory values of the ND and RR groups. Laboratory values for calcium (Ca) were defined as albumin-adjusted Ca values. Laboratory Ca values, hemoglobin (Hb) levels, and the free light chain (FLC) ratio were significantly different between the ND and RR groups, but there was no difference between the two groups in the other laboratory values.

Changes in serum elemental concentrations

The serum concentrations of each element in the control and MM groups are shown in Table 3 . Significant differences between the control and MM groups were observed for Cr, Cu, Mo, and Ba. The changes in concentration of each element in the ND and RR groups were analyzed, but no elements had significantly different serum levels. However, although the value was not significant for Cu, there was a tendency for it to be higher in the RR group than in the ND group (P = 0.060). The Cu/Zn ratio is shown in Table 3 . There was no significant difference between the control and MM groups.

Correlation between serum element concentrations and various laboratory values

Figure 1 shows the correlations between serum Cr, Cu, Mo, and Ba concentrations and laboratory values related to disease state and tumor volume in patients with MM. Although the ND and RR groups consisted of the same patients, they were treated as separate cases because of the different tumor statuses, and the correlation was analyzed as though there were 24 patients. Due to lack of data, β2-microglobulin (β2MG) was analyzed in 19 patients, the FLC ratio in14, urine protein (UP) in 15, and the ratio of plasma cells in bone marrow in 18.

Cr was negatively correlated with serum creatinine and UP levels (P < 0.001 and P < 0.05, respectively) and positively correlated with the estimated glomerular filtration rate and total protein (TP) levels (P < 0.01 and P < 0.05, respectively). Cu was negatively correlated with the TP level (P < 0.01). There were no significant correlations between Mo concentration and any laboratory values. Ba concentration was positively correlated with Hb level (P < 0.01).

Discussion

The present study is the first to comprehensively measure element concentrations in MM patients and investigate their relationship with pathological conditions. Moreover, it is rare to be able to analyze specimens from the same patient at both diagnosis and relapse, which makes this study unique and significant. The results showed that serum Cr, Cu, Mo, and Ba concentrations were significantly increased in MM patients compared to non-MM volunteers. Changes in serum concentrations of these elements may reflect the pathological condition of MM.

Ba is taken up from the plasma by the bone, and about 90% of the body’s Ba is contained in the bone (World Health Organization, 1990; Kovrlija et al., 2021). Most MM patients show generalized osteopenia and osteolytic lesions during the course of the disease, and approximately 60% of MM patients develop pathological fractures (Melton et al., 2005; Silbermann and Roodman, 2013; Hiasa et al., 2021). In our study, 10 of the 12 patients in the MM group had bone lesions at diagnosis. Therefore, our results suggest that the concentration of Ba in MM may be a marker for the pathological condition, particularly reflecting bone lesions of MM. In this study, the association between the presence or absence or number and size of bone lesions and Ba concentration could not be evaluated because most of the patients in the MM group already had bone lesions at the time of diagnosis. Consequently, there may have been no significant difference in Ba concentration between the ND and RR groups. There were two patients who did not have clear bone lesions throughout the course of their disease and had Ba concentrations of 1.44 µg/L and 1.87 µg/L at diagnosis and 1.28 µg/L and 2.24 µg/L at relapse, respectively, which were not lower than those of patients with bone lesions. Another two patients had bone lesions at diagnosis but no obvious bone lesions at relapse. In one patient, the Ba increased from diagnosis to relapse (from 1.74 µg/L to 2.13 µg/L), whereas in the other, the Ba concentration decreased (from 2.15 µg/L to 1.60 µg/L). Although there was no consistent trend in the ND and RR groups, the serum Ba concentration was significantly higher in the MM group than in the control group. These results suggest that higher serum Ba concentrations may be related to bone lesions that were not detected by CT or MRI; however, a larger sample size is needed to confirm this. Nevertheless, being able to determine the exact time to start treatment based on the serum Ba concentration would be extremely beneficial.

Ba, like Ca, is an alkaline earth element. Ca is a major component of the bone. In the present case, two patients at diagnosis and three at relapse received bisphosphonates or elcatonin, and serum Ca concentrations were decreased by the drugs. Hypercalcemia is a major manifestation of MM that is often treated by medication as, it causes clinical symptoms that affect quality of life, such as nausea, vomiting, and anorexia. It is speculated that the administration of such medications is one of the reasons why laboratory Ca values were lower in the RR group than in the ND group. The mechanism by which Ba accumulates in the bone remains unclear. It has been reported that Ba 2+ has a larger ionic radius than Ca 2+ and is more likely to be eliminated during the recrystallization process of hydroxyapatite (Bligh and Taylor, 1963). Therefore, Ba is likely deposited in the bone by another mechanism. It is possible that Ba is adsorbed on the bone surface in the form of colloidal particles by reaction with phosphate groups (Bligh and Taylor, 1963; Kovrlija et al., 2021). This difference in distribution in the bone may be why we observed a difference in the serum Ba concentration, but not in the Ca concentration, between the control and MM groups.

Cu is an essential element and is involved in a variety of physiological actions as a cofactor for many enzymes, including oxidoreductases (Tsang et al., 2021). It has been shown that Cu is involved in tumor initiation, growth, and metastasis (Cheng et al., 2022). A study from Iran reported increased serum Cu concentrations and decreased serum Zn concentrations in patients with stage Ⅰ MM (Khadem-Ansari et al., 2018). Cu and Zn are part of the active site of superoxide dismutase (SOD) (Banks and Andersen, 2019). Khadem-Ansari et al. (2018) also reported a decrease in Cu/Zn SOD activity in patients with stage Ⅰ MM. In our study, no significant changes in Zn concentration or the Cu/Zn ratio were observed in patients with MM, but an increase in Cu concentration was observed. Cu concentrations were higher in the RR group than in the ND group, but this was not significant. This trend suggests that Cu can be useful as a marker for relapse. It is not clear why no change in Zn concentration was observed in our study. One of the factors may be that our study included patients with various stages of disease. Future studies should examine larger cohorts.

Cr has been reported to be carcinogenic (Kawanishi et al., 1986; Nickens et al., 2010; Balali-Mood et al., 2021). In occupationally exposed individuals, an increased risk of developing various malignancies has been reported (Hara et al., 2010; Deng et al., 2019). In the present study, no patients had information on exposure to high concentrations of Cr. No previous studies have examined the mechanism by which increased Cr is associated with MM; changes in Cr concentration may be due to MM symptoms or changes in the general condition.

Mo is an essential element in humans and is present in the active sites of enzymes such as xanthine oxidase, sulfite oxidase, and aldehyde oxidase (Komada et al., 1990; Forrer et al., 2001). The reason for the elevated Mo concentration in MM patients in this study was not clear, and no prior reports have examined this, but this, increase in Mo may reflect changes in pathological condition and systemic status in MM.

This study had several limitations. It was a small pilot study of 12 patients with MM, and a larger prospective observational study that includes presence and extent of bone lesions and high and low tumor volume would be needed to investigate the roles of changes in serum element concentrations in the pathogenesis of MM, especially in accurately assessing the association between Ba and bone lesions. In addition, for the control group, a large group of individuals without MM should be established for which detailed information, including laboratory values, can be collected. The relationship between the pathological condition of MM and trace elements may be further clarified by examining patients with various stages of MM, such as the remission stage after primary treatment for MM. However, no previous studies examining this relationship exist, and this study may provide an opportunity to examine this relationship.

In conclusion, we found that serum Cr, Cu, Mo, and Ba concentrations were significantly increased in MM patients compared to non-MM volunteers, suggesting a relationship between the pathological condition of MM and elemental concentrations. Future studies with large sample sizes are needed to investigate this trend.

Author Contribution Statement

Y.Y., M.K., Y.H., Y.T., M.R., H.K., and K.K. were involved in the conception and design of the study. Y.Y. and M.K. obtained the data. M.K and Y.T. were involved in participant recruitment. Y.Y. drafted the manuscript. M.K., Y.H., Y.T., A.S., T.K., Y.F., M.R., H.K., S.I. and K.K. revised the manuscript. All authors read the manuscript and approved the final version.

Acknowledgements

Not applicable.

Funding statement

This study did not rely on any external sources of funding.

Student Thesis

Not applicable

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Ethics approval

This study was approved by the Ethics Committee of Nagoya City University Graduate School of Medical Sciences (60-00-1118) and the Epidemiological Ethics Review Committee of the Aichi Prefectural Institute of Public Health (18E-07). Informed consent was obtained from all study participants in accordance with the Helsinki Declaration.

Availability of data

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

Azarm T Fazilati M Azarm H Azarm A Serum selenium levels in chronic lymphocytic leukemia Adv Biomed Res 2013 2 44 24516844 10.4103/2277-9175.114177 PMC3905346

Balali-Mood M Naseri K Tahergorabi Z Khazdair MR Sadeghi M Toxic mechanisms of five heavy metals: Mercury, lead, chromium, cadmium, and arsenic Front Pharmacol 2021 12 643972 33927623 10.3389/fphar.2021.643972 PMC8078867

Banks CJ Andersen JL Mechanisms of SOD1 regulation by post-translational modifications Redox Biol 2019 26 101270 31344643 10.1016/j.redox.2019.101270 PMC6658992

Bligh PH Taylor DM Comparative studies of the metabolism of strontium and barium in the rat Biochem J 1963 87 612 8 13971638 10.1042/bj0870612 PMC1202008

Camiolo G Barbato A Giallongo C Iron regulates myeloma cell/macrophage interaction and drives resistance to bortezomib Redox Biol 2020 36 101611 32863212 10.1016/j.redox.2020.101611 PMC7327252

Campanella A Santambrogio P Fontana F Iron increases the susceptibility of multiple myeloma cells to bortezomib Haematologica 2013 98 971 9 23242599 10.3324/haematol.2012.074872 PMC3669455

Cheng F Peng G Lu Y Relationship between copper and immunity: The potential role of copper in tumor immunity Front Oncol 2022 12 1019153 36419894 10.3389/fonc.2022.1019153 PMC9676660

Deng Y Wang M Tian T The effect of hexavalent chromium on the incidence and mortality of human cancers: meta-analysis based on published epidemiological cohort studies Front Oncol 2019 9 24 30778374 10.3389/fonc.2019.00024 PMC6369173

Demir C Demir H Esen R Altered serum levels of elements in acute leukemia cases in Turkey Asian Pac J Cancer Prev 2011 12 3471 4 22471499

Forrer R Gautschi K Lutz H Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS Biol Trace Elem Res 2001 80 77 93 11393312 10.1385/BTER:80:1:77

Greipp PR San Miguel J Durie BG International staging system for multiple myeloma J Clin Oncol 2005 23 3412 20 15809451 10.1200/JCO.2005.04.242

Hara T Hoshuyama T Takahashi K Delgermaa V Sorahan T Cancer risk among Japanese chromium platers, 1976-2003 Scand J Work Environ Health 2010 36 216 21 20024521 10.5271/sjweh.2889

Hiasa M Harada T Tanaka E Abe M Pathogenesis and treatment of multiple myeloma bone disease Jpn Dent Sci Rev 2021 57 164 73 34611468 10.1016/j.jdsr.2021.08.006 PMC8477206

Kanda Y Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics Bone Marrow Transplant 2013 48 452 8 23208313 10.1038/bmt.2012.244 PMC3590441

Kawanishi S Inoue S Sano S Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide J Biol Chem 1986 261 5952 8 3009436

Kazandjian D Multiple myeloma epidemiology and survival: A unique malignancy Semin Oncol 2016 43 676 81 28061985 10.1053/j.seminoncol.2016.11.004 PMC5283695

Khadem-Ansari MH Asoudeh M Gheshlaghi HFK Copper and zinc in stage I multiple myeloma: relation with ceruloplasmin, lipid peroxidation, and superoxide dismutase activity Horm Mol Biol Clin Investig 2018 37 10.1515/hmbci-2018-0055 30367794

Komada H Kise Y Nakagawa M Effect of dietary molybdenum on esophageal carcinogenesis in rats induced by N-methyl-N-benzylnitrosamine Cancer Res 1990 50 2418 22 2317826

Kovrlija I Locs J Loca D Incorporation of barium ions into biomaterials: dangerous liaison or potential revolution? Materials (Basel) 2021 14 5772 34640168 10.3390/ma14195772 PMC8510018

Li T Shi L Wei W Xu J Liu Q The trace that is valuable: serum copper and copper to zinc ratio for survival prediction in younger patients with newly diagnosed acute myeloid leukaemia BMC Cancer 2023 23 14 36604732 10.1186/s12885-022-10486-7 PMC9817254

Mehri A Trace elements in human nutrition (II) - An update Int J Prev Med 2020 11 2 32042399 10.4103/ijpvm.IJPVM_48_19 PMC6993532

Melton LJ Kyle RA Achenbach SJ Oberg AL Rajkumar SV Fracture risk with multiple myeloma: a population-based study J Bone Miner Res 2005 20 487 93 15746994 10.1359/JBMR.041131

Nickens KP Patierno SR Ceryak S Chromium genotoxicity: A double-edged sword Chem Biol Interact 2010 188 276 88 20430016 10.1016/j.cbi.2010.04.018 PMC2942955

Nishito Y Kambe T Absorption mechanisms of iron, copper, and zinc: an overview J Nutr Sci Vitaminol (Tokyo) 2018 64 1 7 29491267 10.3177/jnsv.64.1

Shen C Zhang K Yang J Association between metal(loid)s in serum and leukemia: a systematic review and meta-analysis J Environ Health Sci Eng 2023 21 201 13 37159736 10.1007/s40201-023-00853-2 PMC10163180

Siegel RL Miller KD Jemal A Cancer statistics, 2020 CA Cancer J Clin 2020 70 7 30 31912902 10.3322/caac.21590

Silbermann R Roodman GD Myeloma bone disease: Pathophysiology and management J Bone Oncol 2013 2 59 69 26909272 10.1016/j.jbo.2013.04.001 PMC4723362

Stevens J Waters R Sieniawska C Serum selenium concentration at diagnosis and outcome in patients with haematological malignancies Br J Haematol 2011 154 448 56 21770918 10.1111/j.1365-2141.2011.08744.x

Tsang T Davis CI Brady DC Copper biology Curr Biol 2021 31 421 7 10.1016/j.cub.2021.03.054 33974864

VanderWall K Daniels-Wells TR Penichet M Lichtenstein A Iron in multiple myeloma Crit Rev Oncog 2013 18 449 61 23879589 10.1615/critrevoncog.2013007934 PMC3741338

Wada O What are trace elements? -Their deficiency and excess states Jpn Med Assoc 2004 47 351 8

Yuan S Mason AM Carter P Selenium and cancer risk: Wide-angled Mendelian randomization analysis Int J Cancer 2022 150 1134 40 34910310 10.1002/ijc.33902 PMC7613914

Zhang F Zhuang J Pathophysiology and therapeutic advances in myeloma bone disease Chronic Dis Transl Med 2022 8 264 70 36420171 10.1002/cdt3.35 PMC9676126