Natural element standard solutions (1000 µg/ml) of Al, Cd, Cr, Co, Cu, Fe, Pb, Mg, Mn, Ni, Se, and Zn were obtained from Merck, USA. The standard solutions were kept at 4 oC in dark room to reduce the risk of contamination. All works were carried out under clear room condition. High pure nitric acid, perchloric acid and hydrochloric acid, Ultra pure de-ionized water ( 18 M ohm) used were used for sample preparation. Standard solutions were prepared freshly from the stocks, with dilute nitric acid (3% v/v). In order to obtain appropriate ICP-AES responses, the experiments were performed using different concentration levels. Instrument was calibrated using aqueous standards of elements 10, 20, 30, and 40 μg/L. Accuracy and precision of the method for elements were checked by spiking the samples with known amount of standard.

Inductively coupled plasma spectrometer has been extensively used in the analysis of major, minor and trace elements in biological material because of its high sensitivity, accuracy, low matrix effect and simpler operation. The ultra-trace elements in such biological fluids could be determined by ICP – AES [12]. A comparative study on the analytical performance of flame atomic absorption, flame atomic emission and inductively coupled plasma (ICP) emission for biological strontium assays, especially in blood serum was carried out [11]. The present work was performed using ISA JOBIN YNON – 24.

Operating conditions were

R.F Generator : 40.68 MHz, 1000 Watts

Power required : 220 ± 10 V, 50/60 Hz, Single phase 5 kV

Flame Temp : 11000 K

Plasma : Argon

Spectra range : 189 – 800 nm

Sensitivity : ppb level of detection (±.002 ppm)

Presence of various elements in the sample was identified by determining the wave length of the emitted radiation (Al: 394.401 nm, Cd: 226.502 nm, Co: 236.379 nm, Cr: 284.325, Cu: 324.754 nm, Fe: 238.204 nm, Mg: 383.826, Mn: 293.930 nm, Ni: 231.604 nm, Pb:220.353 nm, Se: 196.090 nm, Zn: 213.856 nm) and their concentrations can be calculated by intensity of radiation, which may be sufficiently low for certain applications with a simple matrix. The commercially available liquid chromatography (LC) system was directly connected to the pheumatic nebulizer. All samples were injected directly without any dilution through the 100 µl injection loop while deionized water continuously flowed at 1 ml min-1 via LC pump. The emitted light from the ICP was collected by the monochromator and polychromator simultaneously. The dynamic viscosity of the sample is measured by fluid spectrometer, which requires 1 to 2 ml of digested sample. The temperature was set at 20o C with the stability of ±0.5o C. Standard solutions were prepared by diluting single element solution (1000 mg/L) in the matrix matching solution. Data acquisition and processing were performed using ICP JY version 5.2 software.

1 ml of sample was transferred to a Teflon beaker and 7.5 ml of concentrated nitric acid, 2.5 ml of concentrated percholoric acid were added [14], then brought out very slowly to boiling on a hot plate and heated to dryness. If blackening of samples occurs during the fuming stage, nitric acid was added drop wise then the sample was cooled, 10 ml of deionized water and a drop of concentrated HCl was added. Then the solution was made up to 100 ml with addition of deionized water and analyzed against calibration curve established.

The results of monitoring gallstone patient’s serum metal level before and after surgery are described in terms of mean, standard deviation (SD), and statistically compared using paired sample t test at 95% confident level. p value less than or equal to 0.05 is considered as significant. The statistical package used was SPSS for windows 12.0 version (SPSS, Chicago, IL,USA).

All blood serum /plasma samples of 15 patients 48h before and after gallstone surgery were analyzed using ICP-AES. From the results, serum Al level (Pre 0.0438±0.0252 ppm, post 0.0217 ± 0.0132 ppm), Cd level (Pre 0.0058 ± 0.0021 ppm, post 0.0022 ± 0.0011 ppm); Co level (Pre 0.0176 ± 0.0045 ppm post 0.0060 ± 0.0020 ppm); Cr level (Pre 0.0177 ± 0.0083 ppm, post 0.0061 ± 0.0037 ppm); Cu level (Pre 0.5683 ± 0.0735 ppm, post 0.4663 ± 0.0503 ppm); Fe level (Pre 0.6491 ± 0.1405 ppm, post 0.9396 ± 0.3509 ppm); Mg level (Pre 8.2880 ± 1.1033 ppm, post 6.6320 ± 1.1657 ppm); Mn level (Pre 0.0208 ± 0.0104 ppm, post 0.0061 ± 0.0031 ppm); Ni level (Pre 0.0400 ± 0.0310 ppm, post 0.0925 ± 0.0374 ppm); Pb level (Pre 0.0529± 0.0131 ppm, post 0.0269 ± 0.0105 ppm); Se level (Pre 0.2220 ± 0.0624 ppm, post 0.2971 ± 0.0384 ppm) and Zn level (Pre 0.4407 ± 0.1102 ppm, post 0.3731 ± 0.1126 ppm) were obtained. Fig.1 reports elemental concentrations serum before and after 48h of gallstone surgery.

Figure 1

Table 1. Changes in trace element levels in patients (n=15) at 48h before and after surgery

Figure 2

Fig 1. Changes in trace element levels in patients (n=15) at 48h before and after surgery

Both analytical techniques developed, ICP - AES and ICP – MS, proved fully satisfactory for the purpose of present investigation. Many minerals and trace elements are required as cofactors, coenzymes or metalloenzymes in metabolic pathways and participated in biological functions, oxygen transport, free radical scavenging, structural organization of macromolecular and hormonal activity. The essential function of trace elements is maintaining the physiological condition in many organs.

The elemental concentration of Al, Cu, Cd, Cr, Co, Pb, Mg, Mn and Zn in pre operative serum was higher than the post operative serum concentrations, whereas elevated serum Fe, Ni and Se concentrations were found in 48h after surgery.

Patients undergoing surgery are at risk of increased oxidative stress. The trauma of a surgical procedure is known to promote a pro-oxidative state, due to ischaemia – reperfusion processes [15]. Many of these associations have been documented in various forms of liver disease in other species, including the elevation of copper and decrease in zinc and their concurrent association with elevations of ALT and glutamic pyruvic transaminase (GGT) or decreases in albumin. Cu is an essential trace element for blood formation, most concentrated in the liver, heart , kidneys, brain, bones and muscles and is essential to blood. Statistically significant decrease in Cu concentration was found in post operative patient’s serum. Cu in excess is not common because only a small percentage is assimilated and in those rare instances can present in diseased stages. An elevated level of serum Cu is documented in liver diseases like cirrhosis, obstructive jaundice and cholestasis. Decrease in post operative serum Cu concentration maybe due to the formation of new blood.

Elements such as Fe and Se are needed to maintain biological activity. Fe is necessary for healthy blood chemistry and is essential for recovery from illness. Without sufficient Fe body cannot manufacture enough new hemoglobin. The statistically significant increase in serum Fe concentration was found in post operative patients. The increase in serum Fe level is due to damage of erythrocytes in the extracorporeal circuit damage not evident as visible hemolysis but sufficient to cause an increases in serum Fe [16]. Fe is also needed for proper muscle and organ function. Fe from these sources is readily absorbed in the intestines. The increase in serum Se reduces the cancer mortality. Low level of Se imports liver necrosis which causes jaundice. Pigmented stones are mainly due to hemolysis. Serum Zn concentration is not statistically significant in the study. More than 300 enzymes in the human body requires Zn for proper functioning that is vital to many biological functions such as immune resistance, wound healing, digestion, reproduction, physical growth etc. The decrease in serum Zn may also be related to redistribution of Zn to the site of tissue injury. It has been shown that radio labeled Zn localizes in actively healing wounds, reaching maximal concentration in tissue within 24 to 48h after injury [17] and nonspecific reaction to stress [16]. Sixty percentage of Cu in the blood is tightly bound to a Cu-Zn-dependent enzyme known as superoxide dismutase (CuZnSOD) which is a powerful anti- oxidant enzyme. A similar antioxidative enzyme is dependent on the trace mineral manganese (MnSOD). As with glutathione peroxidases(GSH-Px) the role of superoxide dismutases is to protect cells against free-radical injury [18]. Addition of single or multiple trace elements as antioxidants (specifically Se) during parental nutrition in human studies was associated with a significant decrease in mortality. Se is a vital component of GSH-Px which acts as a powerful antioxidant enzyme. The patient recovering from surgery may be in a state of negative anti-oxidant balance, which can only be corrected by nutritional input. Se with other trace elements play a role in disease-condition. The highest GSP-Px activity has been found to occur in the liver, moderately high in erythrocytes, heart muscle, lung and kidneys, and lesser in the intestinal tract and skeletal muscles [19]. Decrease in Cr is linked with various effects and diseases like arteriosclerosis, ischaemic heart disease, lipid metabolism, hyperglycaemia and protein synthesis. Some of the trace elements fall in the group of non-essential or toxic (Pb, Cd, Hg, Al, Co, Cr) trace elements because their biological significance is confined and the change is mainly due to environmental factors. Antioxidants such as MnSOD can neutralize free radicals and may reduce or even help to prevent some of the damage of infected body organ. Mn acts as a catalyst and cofactor in many enzymatic processes involved in the synthesis of fatty acids and cholesterol. Low levels of Mn have also been associated with muscle disorders. Several minerals, such as Ca, Fe and possibly Zn can reduce the absorption of Mn. Cu is needed to help the body to use iron whereas Mg is used by the body to maintain muscles, nerves, and bones. Mn is required to manufacture enzymes necessary for the metabolism of proteins and fat. Lower hepatic Cr is reported diabetic patients. Decrease in serum Cr maybe associated with temporary body metabolic disorder. Cr is essential for maintaining the structural stability of proteins and nucleic acids and studies on animals have found that this element is also vital for healthy foetal growth and development. Change in serum trace elements like Cd, Zn concentration may associate with elevated Se and Fe respectively. Increase in Fe is also linked with a decrease in Mn. Mn also supports the immune system, blood sugar balance, and is involved in the production of cellular energy, reproduction and bone growth. Closely associated with Cu, Zn and Mn. Low levels of Mn in blood or tissue have been associated with several chronic diseases and muscle disorders. Mn is a natural tranquilizer. Change in serum Mn level due to loss of antioxidant property of blood in cardiac heart disease and carcinogenesis [20] Changes in other elemental concentrations are not known that may also be the reason of malnutrition, it disturbs cellular and organ functions.

We thank AN. Kannappan, Professor and Head, Department of Physics, Annamalai University for providing necessary facilities. We also thank Dr. R.Baskaran and other members of Department of Surgery (RMMC&H) Annamalai University for their help in this work.