Changes in es­sential and trace elements content in degenerat­­ing human intervertebral discs do not cor­respond to patients’ clinical status

Overview

Aim: To date, there has been no paper consider­­ing the disc degeneration process in respect of the content of es­sential and trace elements in degenerat­­ing discs tis­sue, clinical status of patients, and imag­­ing analysis. Concentration of es­sential and trace elements in disc tis­sue may be a consequence of both environmental and genetic factors. The study aims to analyse and assess the contents of essential and trace elements in intervertebral discs.

Patients and methods: The material of 19 intervertebral discs was obtained from 17 patients dur­­ing lumbar discectomy. Control was 9 healthy discs obtained from organ donors. Atomic absorption spectrometry was used to as­sess levels of Cu, Fe, Mn, Pb, Zn, Na, Mg, K, Ca, and P in the tis­sue, as well as dry weight (d.w.) of the tis­sue.

Results: All 10 es­sential and trace elements were detected in all samples. A significant increase of Ca, Mg, Fe, and P, and decrease of Cu and K in operated discs was found; the remain­­ing changes between unhealthy and healthy discs were not significant. There were no age /  elements, Pfir­rmann grade /  elements, or Modic grade changes /  elements cor­relations. A significant positive cor­relation was found between Mg and Zn, K and Fe, Ca and Zn, Ca and Mg, P and Zn, P and Mg, and P and Ca. A negative cor­relation was only indicated between age and Na. Ca levels were higher in the degenerat­­ing disc group than in the healthy group.

Conclusion: A lack of cor­relation between the Ca content and the stage of intervertebral disc degeneration as well as the age of patients in the degenerat­­ing disc group is an unexpected result.

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人椎间盘退变过程中必需元素和微量元素含量的变化与患者的临床情况并不相符

目的:

到目前为止，还没有文献考虑椎间盘退变过程中退变组织中必需和微量元素的含量、患者的临床状况以及影像学分析。椎间盘组织中必需和微量元素的富集可能是环境和遗传因素共同作用的结果。本研究旨在分析和评价椎间盘中必需和微量元素的含量。

患者和方法:

对17例腰椎间盘摘除术患者的19个椎间盘材料进行分析。对照组为9例来自器官捐赠者的健康椎间盘。采用原子吸收光谱法测定组织中铜、铁、锰、铅、锌、钠、镁、钾、钙、磷的含量，以及组织的干重(d.w.)。

结果:

所有样品中均检出10种必需及微量元素。手术椎间盘中Ca、Mg、Fe、P明显升高，Cu、K明显降低;不健康和健康椎间盘之间的剩余变化不显著。年龄/因素、Pfirrmann评分/因素、Modic评分变化/因素之间无相关性。Mg与Zn、K与Fe、Ca与Zn、Ca与Mg、P与Zn、P与Mg、P与Ca呈显著正相关，仅年龄与Na呈负相关。椎间盘退变组Ca水平高于正常组。

结论:

Ca含量与椎间盘退变的分期及退变椎间盘组患者的年龄之间缺乏相关性是一个出乎意料的结果。

关键词:

椎间盘退变-必需和微量元素-腰椎间盘疾病

Keywords:

intervertebral disc degeneration – essential and trace elements – lumbar disc disease

Introduction

The process of degeneration of human intervertebral discs is a problem which has been investigated in a number of ways, and yet there are still many elements which remain unexplained. Many putative factors concern­­ing its aetiology have been suggested, such as genetics, which seem the most important, as well as causes of an environmental origin [1– 5].

The trigger point in disc degeneration is an injury of the disc, and the changes fol­low­­ing the injury are aber­rant cel­l-mediated responses to the structural damage [1].

Factors like age, genetic inheritance, and a history of inadequate transport and load­­ing of metabolites can weaken discs to such an extent that structural failure occurs dur­­ing daily activities such as physical ef­fort, or even sneez­­ing or coughing [1,2].

At the moment, we are faced with the chicken and egg scenario –  which came first, the chicken or the egg? Are the structural changes a cause or an ef­fect of the disc degeneration proces­s?

Many factors and substances were examined in intervertebral discs to answer the fol­low­­ing questions: 1. what is disc degeneration; 2. what triggers it, and 3. how does the process continue.

A degenerat­­ing intervertebral disc undergoes many changes which are dif­ferent in nature, such as bio­mechanical (structural damage) or bio­chemical (levels of many complex substances as well as chemical elements in the tis­sue are changing).

In this paper, the author would like to focus attention on the chemical changes in degenerat­­ing intervertebral discs, and the relationship of these changes to the clinical status of the patients.

Exces­sive deposition of selected elements in an avascular adult intervertebral disc can lead to the acceleration of a cascade of adverse metabolic changes that normal­ly occur dur­­ing ageing; such a situation reflects negatively the stability of the intercel­lular matrix of disc pulpose.

To date, the cor­relations between changes in the contents of selected elements in the intervertebral discs and the clinical patients’ status have not been studied. Such examination might provide more light on the pathogenesis of the disc degeneration process and perhaps newer therapeutic approaches.

The first aim of this study was an evaluation of the dif­ferences of the es­sential and trace elements concentration in intervertebral disc tis­sue between healthy people and patients with degenerative changes. We decided to analyse (accord­­ing to the other authors [6,7]) the fol­low­­ing elements: Cu, Fe, Mn, Pb, Zn, Na, Mg, K, Ca, and P.

The second purpose of our study was to investigate the cor­relation of the content of these elements with clinical characteristics of the patients with intervertebral disc degeneration.

Patients and methods

The study was approved by the Bioethics Com­mittee of the Medical University of Silesia (decision number KNW/  0022/ KB/ 42/ 15). Nineteen specimens were obtained from 17 patients (7 women, mean age 41.7 years) dur­­ing lumbar discectomies. All specimens were prolapsed lumbar intervertebral disc. All patients matched two basic criteria: 1. discectomy was their first spinal surgery; 2. standard pre-operational 2-month pharmacother­apy was not ef­fective.  Sixteen samples were col­lected from the left site, and three from the right side. Seven specimens were obtained from L5/ S1, eleven  from L4/ L5, and one from L3/ L4 level. All patients were examined and interviewed; data regard­­ing age, gender, level and site of operation, as well as intensity of the pain measured by means of Visual Analogue Scale (VAS) [5,8] were col­lected and are presented in Tab. 1.

Before surgery, all patients underwent a MRI examination. In this examination, each intervertebral disc and vertebral body were analysed in terms of degeneration stage (Pfir­rmann grade) [9], level of surgery, and Modic type endplate changes in adjacent vertebral bodies [10] (Tab. 2). 

The control group was constituted of nine intervertebral discs col­lected from three patients dia­gnosed with brain death, who were refer­red to be organ donors. All these discs were then evaluated for the presence of degenerative changes by two qualified board--certified pathologists. Only completely healthy discs, with no degenerative changes, were included as controls in the research.

Laboratory analyses

Im­mediately after surgery, specimens were deep frozen at – 80 °C and stored. All samples of intervertebral discs were determined in terms of: dry matter (d. m.) and contents of Cu, Fe, Mn, Pb, Zn, Na, Mg, K, Ca and P.

The d. m. content of analysed discs was determined by weigh­­ing a sample before and after complete dry­­ing at 105 °C. On the basis of the dif­ference in sample mass before and after drying, the percentage of dry mass was calculated.

In order to determine the content of elements, material was digested us­­ing the wet method in a closed system in a microwave oven (Multiwave 3000, Anton Paar, Graz, Austria). About 0.5 g of d. m. of samples was treated with a 7 cm³ mixture (1 : 6 v/ v) of concentrated acids, HCl and HNO₃ (Suprapure, Merck, Darmstadt, Germany). Digestion was conducted in teflon ves­sels with maximum power of the oven (1,400 W) for 25 min. The concentration of elements in the obtained filtrate was determined with the use of a PerkinElmer Optima 7300 DV (PerkinElmer, Inc., Waltham, MA, USA), inductively coupled plasma optical emis­sion spectrometer. Each analysis was repeated to ensure the accuracy of the result. If the replication analyses results dif­fered from one another by more than 5%, another two analyses were conducted for that same sample. The results were given in mg × kg^– 1 d. m.

Statistical analysis

Data were analysed us­­ing the Statistica 8.0. computer software (StatSoft, Inc., Tulsa, OK, USA). All variables were tested for normality of distribution us­­ing the Shapiro-Wilk test. Statistical analysis was conducted us­­ing the nonparametric Kruskal-Wal­lis /  Man­n-Whitney U test with post-hoc Dun­na test as well as the analysis of variance for parametric data (Analysis of Variance [ANOVA] test with post-hoc RIR Tukey test). The cor­relation rate was calculated us­­ing the Spearman’s test. The Spearman rank cor­relation coef­ficient was determined. Statistical significance was set at a P value of less than 0.05.

Results

The content of Fe, Zn, Na, Mg, K, Ca, and P in the discs were detected in high levels in all samples. Trace elements (Cu, Mn, Pb) were considered as present when their concentration exceeded 0.6 mg × kg^– 1 d. m.

Mn was detected in 5 (26%) samples, Cu in 8 (42%) samples, and Pb in 4 (21%) samples.

The ranges of concentrations for particular elements were as fol­lows (mean value ± standard deviation, range resp.) (Tab. 3).

Cu mean was 1.19 ± 0.88 mg × kg–1 d.m., range of 0.25–3.19 mg × kg–1 d.m. in degenerating discs, while in the healthy ones the mean was 5.9 ± 1.51 mg × kg–1 d.m., range of 3.5–9.22 mg × kg–1 d.m.

Ca mean was 1.50 ± 1.82% of d.m., range of 0.27–7.69% of d.m. in degenerating disc, while in healthy ones the mean was 0.04 ± 0.02% of d.m., range of 0.02–0.07% of d.m.

Fe mean was 147.42 ± 49.94 mg × kg–1 d.m., range of 68.60–254.30 mg × kg–1 d.m. in degenerating discs, while in healthy ones the mean was 104.57 ± 16.84 mg × kg–1 d.m. range of 75.00–125.60 mg × kg–1 d.m.

K mean was 0.13 ± 0.03% of d.m., range of 0.09 –0.20% of d.m. in degenerating disc, while in healthy ones the mean was 0.30 ± 0.11% of d. m., range of 0.17–0.45% of d.m.

Mg mean was 0.51 ± 0.29% of d.m., range of 0.26–1.40% of d.m., in degenerating disc, while in healthy ones the mean was 0.03 ± 0.01% of d.m., range of 0.26 –1.4% of d.m.

Mn mean was 0.23 ± 0.24 mg × kg–1 d.m. range of 0.15–1.22 mg × kg–1 d.m. in degenerating disc, while in healthy ones the mean was 0.20 ± 0.16 mg × kg–1 d.m., range of 0.15–0.62 mg × kg–1 d.m. 

Na mean was 1.60 ± 0.31% of d.m. range of 0.95–2.16% of d.m. in degenerating disc, while in healthy ones the mean was 1.43 ± 0.20% of d.m., range of 1.08–1.78% of d.m.

P mean was 0.72 ± 0.95% of d.m. range of 0.04–3.98% of d.m., in degenerating disc, while in healthy ones the mean was 0.13 ± 0.03% of d.m., range of 0.08–0.15% of d.m.

Pb mean was 0.83 ± 0.32% of d.m., range of 0.60–1.47% of d.m. in degenerating disc, while in healthy ones the mean was 0.83 ± 0.52% of d.m., range of 0.60 –2.45% of d.m.

Zn mean was 21.90 ± 13.59 mg × kg–1 d.m., range of 7.60–62.20 mg × kg–1 d.m. in degenerating disc, while in healthy ones it was 21.76 ± 13.59 mg × kg–1 d.m.,  range of 7.60–62.20 mg × kg–1 d.m.

The dif­ferences between healthy and degenerat­­ing discs appeared significant for Cu, Fe, Mg, Ca, K and P (Tab. 4).

The levels of K and Cu were higher in healthy discs, while levels of P, Ca, Mg and Fe were higher in degenerat­­ing discs.

In the surgery group, the cor­relation analysis revealed a significant negative relationship between age and sodium content. A positive cor­relation was indicated between VAS and Pfir­rmann grade, Fe and K, Zn and Mg, Cu and P, Na and K, Mg and Ca, Mg and P (Tab. 5).

Discus­sion

Analysis of selected trace elements in degene­rat­­ing intervertebral discs showed a significant increase of Fe, Mg, Ca and P, and a decrease in Cu and K as compared with healthy ones.

Calcium

Calcium is one of 21 es­sential elements for humans [11]; it regulates many intracel­lular and extracel­lular proces­ses. Dysregulation in production, level, or transport of Ca is always as­sociated with a dis­ease. Ca deposits are well known elements of disc degeneration; however, the role of the deposits in the degeneration process is still unclear. Several papers showed that there was a cor­relation between the presence of Ca crystals  and disc degeneration phenomenon [12– 14]. Some data advocate that deposits present in degenerated intervertebral discs are made of calcium pyrophosphate dihydrate, and this phenomenon is more associated with the previous history of trauma or surgery [12]. On the other hand, there are data which state that the calcium pyrophosphate dihydrate is both the cause and ef­fect of disc degeneration [13]. In the literature, there is still a lack of quantitative analysis; therefore, data contained in this paper may be helpful in understand­­ing the process of disc degeneration. In the degenerat­­ing disc group, there was no cor­relation between age and Ca levels, as well as between Ca levels and Pfir­rmann grade of degeneration. Consider­­ing the dif­ferences in Ca concentration between healthy and degenerat­­ing discs, there was a higher Ca content in degenerat­­ing discs than in healthy ones.

Copper

Copper is an active metal characteristic for organisms liv­­ing in an oxygen-rich environment. It is as­sociated with animal proteins involved in reduction-oxygenation proces­ses [3]. Many enzymes harness the changes in the Cu oxidation stage to catalyse redox reactions in a numerous range of bio­chemical transformations [15]. Cu plays an important role in cell haemostasis, and cell signal­l­­ing proces­ses. Moreover, Cu handl­­ing and Cu utilis­­ing proteins control metabolic changes in cancer cel­ls known as the Warburg ef­fect –  the down-regulation of cell respiratory capacity observed in cancer cel­ls [15]. In examined groups, there was a higher concentration of Cu in the healthy group, which may be a consequence of improved oxygenation and improved blood supply to healthy disc tis­sue [4].

Iron

The role of Fe in many proces­ses is dif­ficult to overestimate. As a very important component of haemoglobin, it plays a significant role in oxygen transport. There are four clas­ses of Fe-related proteins: Fe contain­­ing haeme proteins (haemoglobin, myoglobin, cytochromes), iron sulfur enzymes (flavoproteins, haemaflavoproteins), proteins for Fe storage and transport (transfer­rin, lactofer­rin), and other Fe-contain­­ing and Fe-activated enzymes. The role of Fe in the disc degeneration process is still unknown, and there are no papers describ­­ing this topic [16]. The content of Fe was higher in degenerat­­ing discs, and this result was statistical­ly significant.

Sodium and potas­sium

Sodium and potas­sium as well as their attendant anions are important components of all body fluids [17]. Na and K play a principal role in maintain­­ing body fluid homeostasis. Levels of these ions are important for water balance, and disc dehydration is one of the components of disc degeneration. These two ions are important in the creation of nerve impulses, us­­ing concentration gradients across plasma membrane produced by Na(+), K(+) adenosine triphosphate (ATP)-ase [18].

Our results show higher levels of K in healthy discs, and a negative cor­relation between age and the content of Na; both results are statistical­ly significant.

Magnesium

Magnesium is the second most abundant intracel­lular cation, and fourth cation in terms of abundance for the whole body. This cation is es­sential for the synthesis of nucleic acids and proteins, and plays a role in Ca metabolism by compet­­ing with Ca for membrane binding. Mg has many important bio­logical functions, such as intracel­lular energy metabolism cell replication, and protein synthesis [19]. Levels of Mg were higher in degenerat­­ing discs and the dif­ference was statistical­ly significant.

Manganese

Manganese is es­sential for bone formation. It plays an important role in the metabolism of amino acids, lipids, and carbohydrates. Glycosyltransferases and xylotransferases are important in proteoglycan synthesis and they are very sensitive in the presence of Mn. Thus, the latter can play a role in the disc degeneration proces­s [6]. The levels of Mn in healthy discs and degenerat­­ing discs were similar, and the dif­ference was not statistical­ly significant.

Phosphorus

Numerous normal physiologic functions are dependent on P, includ­­ing skeletal develop­ment, cell membrane phospholipid content and function, cell signal­ling, platelet aggregation, and energy transfer through mitochondrial metabolism [6]. P is es­sential for the bone mineralisation proces­s [20]. The level of P was higher in degenerat­­ing discs, and the dif­ference was statistical­ly significant.

Lead

Lead accumulates in bones and its concentration tends to increase with age, because lead is dif­ficult to remove from the tis­sue [21]. More than 90% of the body’s Pb burden is found in the skeleton [8]. The bio­logical half--life of lead is about one month for soft tis­sue, it is longer –  years –  for trabecular bones, and decades for cortical bones [22].  Pb can cause several adverse health ef­fects, such as neuropathy, encephalopathy, and kidney damage. Pb levels in intervertebral discs should not be high, because most Pb cumulates in bones. This is the case in the presented group, where only four specimens showed Pb levels higher than 0.60 mg × kg^–1; the dif­ference between healthy and degenerat­­ing discs was not significant.

Zinc

Zinc is a component of various enzymes; it forms and helps to maintain the structural integrity of proteins and regulates gene expres­sion [16]. The bio­logical role of Zn can be divided into three categories: structural, catalytic, and regulatory. Zn plays a crucial role in the im­mune system, and Zn-deficient individuals present increased susceptibility to infection [23]. The inflam­matory process in disc degeneration is still to be examined and at the moment we know that it is a part of the whole degeneration proces­s [1,24,25]. Moreover, matrix metal­loproteinases are Zn--dependent enzymes, and these enzymes are responsible for extracel­lular matrix synthesis and degradation. Balance between these two proces­ses is a basic condition to stop the degeneration proces­s [1,24]. Zn levels may indirectly indicate a metal­loproteinase concentration and activity in the disc tis­sue. The dif­ference in Zn levels between healthy and degenerat­­ing tis­sue was not statistical­ly significant.

Conclusions

The study is one of only a few to present elements concentration in vertebral disc tis­sue; moreover, there are no papers analys­­ing either a healthy control group or the clinical status of the patients analysed (MRI images and elements contents).

The results show­­ing dif­ferences between healthy and degenerat­­ing discs in terms of Ca levels are particularly important. There is a statistical­ly significant dif­ference between healthy and degenerat­­ing discs (the level of Ca is higher in degenerat­­ing discs),  while there is no cor­relation between Ca levels and the age of the patient, and Ca levels and disc degeneration stage. The examined group may be too small to demonstrate such cor­relation, but if this fact is confirmed upon further examination, questions regard­­ing Ca chemistry and its role in disc degeneration process should be formulated. 

Levels of other elements may be influenced by diet, and other exogenous factors such as contamination which is associated with technological development of the dwel­l­­ing location.

Identification of significant factors as well as their influence on the disc degeneration process are both is­sues still demand­­ing further investigation.

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manu­script met the ICMJE “uniform requirements” for biomedical papers.

Accepted for review: 24. 9. 2018

Accepted for print: 7. 2. 2019

Wiesław Marcol, MD, PhD

Medical University of Silesia

Medyków 18

407 52 Katowice

Poland

e-mail: wmarcol@tlen.pl