The value of copper and zinc levels in hernia
formation
Suleyman Ozdemir*, Erpulat S. Ozis*, Kamil Gulpinar*, Sinan M. Aydın*, Ayse A. Eren†, Selda Demirtas† and Atila Korkmaz*
*
Departments of Surgery,†Biochemistry, Ufuk University Medical School, Ankara, Turkey
ABSTRACT
Background A defect in collagen metabolism is suspected to be one of the factors responsible for hernia formation. Lysyl oxidase is a copper-dependent enzyme in the process that provides for the structural integrity of collagen molecules, while zinc is essential for tissue maintenance.
Materials and methods In a prospective fashion, copper and zinc levels were measured in plasma and tissue specimens obtained from indirect (n = 23), direct (n = 20) and incisional hernia patients (n = 19) and from healthy controls (laparoscopic cholecystectomy patients, n = 15) by enzymatic colorimetric analysis.
Results Groups were similar in age, comorbid diseases and body mass index. Whereas plasma levels of Cu and Zn in hernia and control patients were similar, and tissue levels were significantly lower in all hernia groups (especially the incisional hernia group) compared to controls (P < 0Æ001). The incisional hernia group had signifi-cantly lower tissue copper levels than direct hernia patients and lower zinc levels than indirect hernia patients. Conclusions Patients with all types of hernia, especially those with incisional hernias, have significantly lower tissue copper and zinc levels than control patients, despite having similar plasma levels. This finding might reflect excessive consumption or dysfunction of lysyl oxidase as playing a role in the aetiology of hernias. Keywords Copper, fascia transversalis, hernia, zinc.
Eur J Clin Invest 2011; 41 (3): 285–290
Introduction
Although inguinal hernia is one of the most common medical problems requiring operative repair, its aetiology remains unclear [1]. In the past, an open processus vaginalis or excessive mechanical stress (e.g. increase in intraabdominal pressure) has been widely accepted predisposing factors for groin hernia [2]. Many, but not all, patients with processus vaginalis or
increased intraabdominal pressure will develop an inguinal hernia. Therefore, an additional process, perhaps a connective tissue pathology, may be involved in the genesis of groin her-nias. Similarly, the aetiology of incisional hernia is multifacto-rial, and its prevalence is independent of gender, unlike that of groin hernia [3–5].
Copper and zinc are important trace elements which are widely distributed in the human body. Copper is an essential element of certain metalloenzymes and proteins such as super-oxide dismutase (SOD) and lysyl oxidase. Zinc is the second most common trace element in the human body after iron and is known to be the main component of over 100 metalloen-zymes. As such, zinc plays an important role in wound
healing, biosynthesis and homeostasis of various connective tissues [6].
Any defect in the fascia transversalis may cause a hernia, because collagen is the main component of this structure; many authors have suggested that any defect in collagen metabolism may lead to hernia formation [7–9]. Although the collagen metabolism of the fascia transversalis and rectus sheath has been studied somewhat [10–14], none to date have evaluated tissue copper and zinc levels in the fascia transversalis.
We hypothesized that low plasma or tissue levels of copper and zinc may lead to hernia formation. To this end, we mea-sured copper and zinc in plasma and tissue samples obtained from the fascia transversalis in subjects without a hernia and in patients with direct, indirect, and incisional hernias.
Patients and methods
The study protocol was approved by our university ethics com-mittee and the hospital institutional review board. Reporting of
the study conforms to STROBE along with references to STROBE and the broader EQUATOR guidelines (Simera et al. January 2010 issue of EJCI). Adult patients who presented to our university hospital surgical department between October 2007 and September 2009 with indirect inguinal hernia, direct inguinal hernia, incisional hernia and those without hernias about to undergo laparoscopic cholecystectomy for gallstones (nonhernia control patients) were approached for participation in the study. Patients granting informed consent were included in the study. Patients with a history of long-term steroid or nonsteroidal drug use, cigarette smoking, chronic obstructive pulmonary disease or collagen diseases were excluded. Patients who had received radiotherapy to the abdominal groin region and recurrent hernia cases were also excluded.
Body weight (kg) and height (m) were measured, and the body mass index (BMI) was calculated (kg m)2). BMI values were classified according to the Clinical Guidelines on the Iden-tification, Evaluation and Treatment of Overweight and Obese in Adults – the Evidence Report [15].
A tissue sample of the fascia transversalis was obtained from patients undergoing primary inguinal hernia through the cen-tral area of the posterior wall of inguinal canal, from patients undergoing incisional hernia from the defective fascia in the midline, and from controls from the posterior part of the rectus sheath from the upper trocar insertion site in the midline. The fascia transversalis and rectus sheath samples were immedi-ately transferred to the laboratory on ice where they were weighed and then stored in a freezer at)80 C until analysis.
For analysis, to every gram of tissue, 10 mL of NaHCO3was
added and the mixture was then homogenized in a DIAX 900 model homogenizer (Heidolph Instruments GmbH & Co., Schwabach, Germany). The homogenizer was cleaned with methanol between samples. Copper and zinc levels were mea-sured spectrophotometrically with a Humalyzer 2000analyzer (Hemagen Diagnostics, Inc., Columbia, MD, USA).
Measurement of zinc levels
When serum was mixed with 5-Br-PAPS [2-(5- bromo-2-pyridy-lazo)-5-(N-n-propyl-N-(3-sulfopropyl) amino) phenol], a coloured complex was formed. Absorbance of the complex at 560 nm was measured spectrophotometrically, and zinc levels in the serum samples were calculated based on absorbance of standard dilutions.
Measurement of copper Levels
At a pH of 4Æ7, copper was separated from ceruloplasmin, and then the serum was mixed with DiBr-PAESA (4-(3,5-dibromo-2-pyridylazo)-N-ethyl-N.sulfopropylaniline) to form a coloured compound. Absorbance at 582 nm was measured spectrophoto-metrically, and copper levels in the serum samples were calculated based on the absorbance of standard dilutions.
Statistical analysis
Data analysis was performed by usingSPSSfor Windows,
version 11.5 (SPSS Inc., Chicago, IL, USA). Normality of the distributions of continuous variables was determined by Shapiro Wilk testing. Data are shown as mean ± standard deviation or median (interquartile range, or IQR), where applicable. While the mean differences among groups were evaluated by one-wayANOVA, Kruskal–Wallis testing was used
to compare median values. The Kruskal–Wallis multiple comparison test was used to determine which group with significantly different values was different than the others. Nominal data were compared by chi-square testing. A P-value < 0Æ05 was considered statistically significant.
Results
Seventy-seven consecutive patients (indirect hernia, Group 1, n = 23; direct hernia, Group 2, n = 20; incisional hernia, Group 3, n = 19) undergoing primary inguinal or incisional hernia
Table 1 Demographic characteristics of patients with indirect hernia (Group I), direct hernia (Group II), incisional hernia (Group III) and nonhernia patients undergoing laparoscopic cholecystectomy (healthy controls, Group IV)
Group I (n = 23) Group II (n = 20) Group III (n = 19) Group IV (n = 15) P
Age (years) 51 ± 14 59 ± 16 61 ± 14 48 ± 15 0Æ072
Male⁄ female 23⁄ 0 18⁄ 2 7⁄ 12 8⁄ 7 < 0Æ001
BMI (kg m)2) 28Æ6 ± 1Æ8 27Æ6 ± 2Æ7 29Æ1 ± 3Æ8 27Æ9 ± 1Æ4 0Æ287
Other comorbid disease 6 (26Æ1%) 9 (45Æ0%) 10 (52Æ6%) 5 (33Æ3%) 0Æ308
Hypertension 4 (17Æ4%) 9 (45Æ0%) 10 (52Æ6%) 3 (20Æ0%) 0Æ042
Diabetes mellitus 2 (8Æ7%) 2 (10Æ0%) 3 (15Æ8%) 1 (6Æ7%) 0Æ837
Hyperlipidemia 1 (4Æ3%) 2 (10Æ0%) 2 (10Æ5%) 2 (13Æ3%) 0Æ773
repair agreed to participate in the study. In addition, 15 patients about to undergo laparoscopic cholecystectomy agreed to participate as healthy controls.
Mean ages of the control group and hernia groups were simi-lar (P > 0Æ05, Table 1): Group 1, 50 ± 14 years (range 23–77 years); Group 2, 59 ± 15 years (range 23–80 years); Group 3, 60 ± 13 years (range 25–77 years) and controls, 48 ± 15 years (range 22–75 years). The percentage of men was 100% in Group 1, 90% in Group 2, 37% in Group 3 and 53% in controls. Groups 1 and 2 had a significantly higher proportion of men than Groups 3 and controls (P < 0Æ001 and P < 0Æ001, respectively, Table 1). No differences between the hernia groups and controls regarding BMI and concomitant diseases were found (P = 0Æ287 and P = 0Æ308, respectively).
The median plasma copper level was 70Æ5 (IQR: 18Æ0) lg dL)1 in Group 1, 72Æ5 (IQR: 21Æ9) lg dL)1in Group 2, 83Æ0 (IQR: 34Æ0) lg dL)1in Group 3 and 73Æ0 (IQR: 33Æ0) lg dL)1in controls. The median zinc level was 76Æ5 (IQR: 18Æ7) lg dL)1in Group 1, 76Æ6 (IQR: 18Æ3) lg dL)1in Group 2, 76Æ5 (IQR: 18Æ7) lg dL)1in Group 3 and 83Æ8 (IQR: 26Æ3) lg dL)1in controls. No significant differences in plasma copper or zinc levels were found between any of the hernia groups and the control group (Table 2).
The median tissue copper levels were 2336 (IQR: 2714) lg g)1 in Group 1, 2066 (IQR: 6361) lg g)1in Group 2, 900 (IQR: 1009)
lg g)1in Group 3 and 10 507 (IQR: 18 081) lg g)1in controls. The median tissue copper levels of all the hernia groups were lower than that of the control group (P < 0Æ001, Fig. 1). The median tissue zinc levels were 2700 (IQR: 5180) lg g)1in Group 1, 2340 (IQR: 4299) lg g)1in Group 2, 1004 (IQR: 1758) lg g)1in Group 3 and 6748 (IQR: 5777) lg g)1in controls (Table 2). The median tissue zinc levels of all the hernia groups were also lower than that of the control group (P < 0Æ001, Fig. 2).
The median tissue copper level was lower in Group 3 (incisional hernia group) than in Group 2 (direct hernia group) (P < 0Æ05). The median tissue zinc level was lower in Group 3 (incisional hernia group) than in Group 1 (indirect hernia group) (P < 0Æ05).
Discussion
Despite all the improvements in hernia operative techniques, a high recurrence rate is the norm in most countries [4,5]. There-fore, many studies have been performed to try and elucidate the root cause(s) of hernia to prevent their formation and improve postoperative outcome. To date, most investigations of the aetiology of hernias have focused on one of the several aeti-ologies, including genetic predisposition [2,16], gender and age
40 000 30 000 20 000 10 000 0 –10 000
Group I Group II Group III Group IV
T
issue copper (
μ
g g
–1)
Figure 1 Tissue copper levels in patients with indirect hernia (Group 1), direct hernia (Group 2), incisional hernia (Group 3) and nonhernia patients undergoing laparoscopic cholecystec-tomy (healthy controls, Group 4). Levels in Groups 1, 2 and 3 are significantly lower than those in nonhernia controls (Group 4). 20 000 15 000 10 000 5000 –5000
Group I Group II Group III Group IV 0 T issue zinc ( μ g g –1)
Figure 2 Tissue zinc levels in patients with indirect hernia (Group 1), direct hernia (Group 2), incisional hernia (Group 3) and nonhernia patients undergoing laparoscopic cholecystec-tomy (healthy controls, Group 4). Levels in Groups 1, 2 and 3 are significantly lower than those in nonhernia controls (Group 4).
[2,5,7,17], and variations in collagen type, amount, structure and degradation rate. In the present study, men predominated in the indirect and direct hernia groups, which is in concor-dance with other studies [2,18]. Past literature has shown that a defect on the X chromosome which is associated with low tissue levels and activity of lysyl oxidase may be responsible [19]. In contrast, gender was distributed equally in incisional hernia patients. Ageing has been suggested as a factor in the formation of hernias because the ultrastructure of the fascia transversalis has been shown to change with age [17]. These changes include an increase in collagen cross-linkage which results in diminished extractability [7]. The mean age of our patients was between 48 and 60 years, which is similar to that of Bellon et al. [13] who noted these abnormalities more frequently in patients between 41 and 60 years old.
Regarding the role of collagen amount and ⁄ or collagen sub-types in the formation of hernias, while some authors reported no difference from controls in the ratio of type I to type III colla-gen [7], most have found a decrease in the ratio of type I to type III collagen in hernia patients [4,14,20–22]. A decrease in the ratio of type I to type III collagen below a critical level may alter the mechanical properties of the tissues and result in hernia for-mation when subjected to increased intraabdominal pressure [14].
Hernia formation may be related to changes in the ultrastruc-ture of collagen and ⁄ or the collagen fibrils [17]. In addition, upon investigation of a defect in the hydroxylation of the amino acids proline and lysine, no difference was found in hernia patients in proline hydroxylation in the fascia transversalis, but a small decrease in lysine hydroxylation was detected in the direct hernia group [13].
A defect in intra- and intermolecular collagen cross-linking may also result in hernia formation [7]. A decrease in lysine hydroxylation in the fascia transversalis in direct hernia patients may indicate alterations in collagen cross-linking or
glycosylation of the collagen molecule [13]. As was stated previously like other studies in the past [23], a decrease in lysyl oxidase activity is associated with groin hernia and this may be because of low copper levels (copper is located at the active site of the enzyme) in the hernia sac of direct hernias [18]. These changes in collagen may ultimately affect its interaction with other components of the extracellular matrix and lead to changes in the elastic and mechanical properties of the tissue. For these reasons, research should focus on factors, including copper, effecting the hydroxylation of lysine in collagen. According to the results of this study, in the sera of patients in the three hernia groups, copper and zinc levels were not differ-ent from controls. This finding means that oral intake and digestion of these trace elements were similar and intact in all patient groups.
The final factor that may play a role in the formation of hernias is an increase in the degradation of collagen in hernia patients. An excess of MMP-2, a matrix degradation enzyme, has been found in patients with aortic aneurysm [24]. This may reflect a proteolytic effect in various connective tissues, and in fact, an increase in MMP-2 expression and MMP-2 inhibitors has been found in hernia tissues [13,25].
Lower amounts of copper and zinc in hernia tissues compared to controls may occur by several mechanisms. An increase in MMP-2 enzyme activity can result in the overpro-duction of superoxide radicals in response to inflammatory process. In response, Cu-SOD, with copper at its active enzymatic site, may have been over consumed. In addition, the collagen degradation process resulting in herniation may also be associated with an overconsumption of other metalloen-zymes which contain copper on their active sites. All of these conditions could lead to a decrease in tissue copper levels. Currently, 23 MMP genes have been identified in humans, and most are multidomain proteins called matrixins [26]. The matrix degradation enzyme, MMP-2, contains zinc on its active Table 2 Plasma and tissue levels [median (interquartile range)] of copper and zinc in patients with indirect hernia (Group I), direct hernia (Group II), incisional hernia (Group III) and nonhernia patients undergoing laparoscopic cholecystectomy (healthy controls, Group IV)
Group I Group II Group III Group IV P
Plasma copper (lg dL)1) 70Æ5 (18Æ0) 72Æ5 (21Æ9) 83Æ0 (34Æ0) 73Æ0 (33Æ0) 0Æ475
Plasma zinc (lg dL)1) 70Æ4 (19Æ0) 76Æ6 (18Æ3) 76Æ5 (18Æ7) 83Æ8 (26Æ3) 0Æ286
Tissue copper (lg g)1) 2336 (2714)* 2066 (6361)†,‡ 900 (1009)†,§ 10 507 (18 081)*,‡,§ < 0Æ001
Tissue zinc (lg g)1) 2700 (5180)*,¶ 2340 (4299)‡ 1004 (1758)§,¶ 6748 (5777)*,‡,§ < 0Æ001
*Significant difference between Group I and Group IV (P < 0Æ001).
†
Significant difference between Group II and Group III (P < 0Æ05).
‡
Significant difference between Group II and Group IV (P < 0Æ01).
§
Significant difference between Group III and Group IV (P < 0Æ01).
¶
site. One of these enzymes, Zn-SOD, may be released in response to excess superoxide radicals. An additional increase in the release of other zinc-containing metalloenzymes may result in an overconsumption of Zn in hernia tissues. Important metalloenzymes that contain zinc play a role in the regeneration of the cells and wound healing include timidin kinase, DNA polymerase and RNA polymerase. Several metalloproteases are mainly secreted by neutrophils, macrophages and other inflam-matory response cells, thus inflaminflam-matory processes or other unknown secondary factors may also negatively affect collagen metabolism and accelerate the hernia development.
In the literature, only one study investigated the copper level of the patients with direct and indirect hernia [18]. They found that the levels of copper in both sera and hernia sac were lower in the direct hernia group than in the indirect hernia group. On a related topic, Pascual et al. [10] reported that levels of cyto-kines, such as transforming growth factor beta-1 (TGF b1), which are involved in tissue remodelling as an important modulator of metalloproteinases, were significantly higher in the fascia transversalis of patients with direct hernia. This may explain how overdegradation of metalloenzymes (and thus lower tissue levels of copper and zinc) could be caused by TGFb1-mediated enhanced expression of MMP-2.
Conclusion
(1) This is the first study to evaluate both plasma and tissue levels of copper and zinc in indirect, direct and incisional hernia groups and compare them with levels in a control group.
(2) Finding normal plasma Cu and Zn levels leads us to conclude that inadequate intake or abnormal GI absorption of copper or zinc are not related to hernia formation. An abnormality in the tissues themselves must be suspected to explain tissue levels of copper and zinc being markedly different in hernia vs. nonhernia patients.
(3) Finding tissue levels of copper and zinc in hernia patients markedly lower than controls may be because of excessive consumption of metalloenzymes by superoxide radicals which are produced in response to an inflammatory process. That levels of these elements were lower in the incisional hernia patients compared to the inguinal hernia patients may be evidence that inflammation is greater in incisional than in inguinal hernias.
(4) For the above or unbeknownst reasons, copper deficiency in the fascia transversalis causes misfunctioning of lysyl oxi-dase that leads to defective or inadequate collagen synthesis, or tissue zinc deficiency leads to an inadequate healing response, future solutions to ensure successful hernia repair
may include materials which incorporate one or both of these elements. Efforts to maintain normal tissue levels of these elements may be a useful strategy in preventing hernia formation primarily, as well as in decreasing the recurrence of hernias after their repair.
Address
Department of Surgery, Ufuk University Medical School, 31. Sokak 5 ⁄ 12 Bahcelievler, 06490 Ankara, Turkey (S. Ozdemir, E. S. Ozis, K. Gulpinar, S. M. Aydın, A. Korkmaz); Department of Biochemistry, Ufuk University Medical School, 31.Sokak 5 ⁄ 12 Bahcelievler, 06490 Ankara, Turkey (A. A. Eren, S. Demirtas). Correspondence to:Suleyman Ozdemir, MD, Department of Surgery, Ufuk University Medical School, 31.Sokak 5 ⁄ 12 Bahcelievler, 06490 Ankara, Turkey. Tel.: 0090 533 3232502; fax: 0090 312 2044055; e-mail: drsozdemir@hotmail.com Received 19 April 2010; accepted 16 September 2010 References
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