Over-expression of Zip-13 mRNA in kidney and lung during dietary zinc deficiency in Wistar rats

Zinc is an essential nutrient for all organisms, which is involved in the function of numerous key enzymes in metabolism. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while Zip transporters increase intracellular zinc. Previous studies in our laboratory have shown that Zip-1, ZnT-1, Zip-2 and LIV-1 mRNA are associated with zinc level in established human breast cancer in nude mice model. In this study, six zinc transporters: ZnT-1, ZnT-2, ZnT-4, Zip-1, Zip-8 and Zip-13 were chosen. We aim to determine the relation between zinc transporters and zinc level in kidney and lung of Wistar rats. Eighteen Wistar rats were randomly divided into three groups: normal group, zinc-deficiency group and pair-fed group. After 22 days, the rats were killed and organs samples were taken, then zinc transporters mRNA were detected by RT-PCR. Compared with the normal group, Zip-13 shows an up-regulation (P < 0.05) in zinc-deficiency group both in kidney and lung, and Zip-8 was significantly lower (P < 0.05) in zinc-deficiency group in kidney.     

Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line

The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.     

Parameters of Metabolic Syndrome and Its Relationship with Zincemia and Activities of Superoxide Dismutase and Glutathione Peroxidase in Obese Women

Alterations in antioxidant defense in obese people with metabolic syndrome can contribute to oxidative stress. This study assessed the relationship between the parameters of metabolic syndrome and the zincemia, activity of superoxide dismutase, and glutathione peroxidase enzymes in obese women. Seventy-three premenopausal women, aged between 20 and 50 years, were divided into two groups: case group, composed of obese (n = 37), and control group, composed of no obese (n = 36). Analyses of zinc intake, parameters of metabolic syndrome, plasma, and erythrocyte zinc, and activities of superoxide dismutase and glutathione peroxidase were carried out. The mean values of body mass index of obese women and control group were 34.5 ± 3.4 and 21.7 ± 1.9 kg/m², respectively (p < 0.05). In the study, body mass index, waist circumference, and zinc intake were higher in obese women than control group (p < 0.05). The plasma zinc and activity of superoxide dismutase did not show significant differences between obese and controls (p > 0.05). The values of erythrocyte zinc was 36.4 ± 15.0 μg/gHb and 45.4 ± 14.3 μg/gHb and of glutathione peroxidase was 46.4 ± 19.4 U/gHb and 36.7 ± 13.6 U/gHb in obese women and controls, respectively (p < 0.05). The study shows that there are alterations in biochemical parameters of zinc in obese women, with low zinc concentrations in erythrocytes. Regression analysis demonstrates that the erythrocyte zinc and activity of superoxide dismutase enzyme is influenced by components of the metabolic syndrome, and the plasmatic glucose, body mass index, and waist circumference have a negative correlation with this enzyme.     

The Correlation Among the Dynamic Change of Zn<Superscript>2+</Superscript>, ZnT-1, and Brain-Derived Neurotrophic Factor After Acute Spinal Cord Injury in Rats

Zinc plays an important role in regulating the expression of brain-derived neurotrophic factor (BDNF) and its receptor in nervous system, but the correlation among Zn²⁺, zinc transporter, and BDNF in spinal cord injuries (SCI) is not fully understood. The purpose of this study was to investigate the expression of Zn²⁺, zinc transporter 1 (ZnT-1), and BDNF, as well as their correlation in spinal cord-injured rats. One hundred Wistar male rats were divided into two groups: sham-operated group (as control group) and model group. Spinal cord injury was induced in model groups by hemisection of T9 on the left side. Compared with the control group, the serum zinc levels in SCI model group were significantly decreased after surgery, but zinc concentrations in spinal cord were increased gradually. The mRNA levels of ZnT-1 and BDNF were significantly increased in SCI model group, and there is a positive correlation between them (Spearman rho = 0.381, P = 0.0204). The correlation found between BDNF and ZnT-1 allows us to speculate that these two factors may be physiologically co-regulated, which may provide an idea for the treatment of SCI.     

Regulation of zinc transporters by dietary flaxseed lignan in human breast cancer xenografts

Zinc is essential for cell growth. Previous studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues. Zinc cannot passively diffuse across cell membranes and specific zinc transporter proteins are required. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while ZIP transporters increase intracellular zinc. In this study, three human zinc transporter members: ZnT-1, ZIP2 and LIV-1 were chosen. We aimed to determine the effect of flaxseed lignan on the growth of ER-negative breast cancer cells in a nude mice model and observe the effect of flaxseed lignan on the regulation of the three zinc transporter in mRNA level. Nude mice were xenografted with human breast cancer cell line MDA-MB-231 and 6 weeks later were fed either the basal diet (BD) or BD supplemented with 10% FS and SDG for 5 weeks. The SDG levels were equivalent to the amounts in the 10% FS. RT-PCR was performed. Compared with the BD group, the tumor growth rate was significantly lower (P < 0. 05) in the FS and SDG group. ZnT-1 mRNA level in mammary tumor was increased in SDG group and decreased in FS group, but no significant difference was found. Extremely low amplification of ZIP2 from mRNA was detected, with no difference between the treatment groups. LIV-1 mRNA expression of SDG group increases compared with BD group. In FS group, it significantly increases nearly 9 times than that in BD group (P < 0. 005).     

Trace Elements in Obese Turkish Children

The quality of the diet of obese children is poor. Eating habits may alter micronutrient status in obese patients. In this study, we determined the serum levels of selenium, zinc, vanadium, molybdenum, iron, copper, beryllium, boron, chromium, manganese, cobalt, silver, barium, aluminum, nickel, cadmium, mercury, and lead in obese Turkish children. Thirty-four obese and 33 healthy control subjects were enrolled in the study. Serum vanadium and cobalt levels of obese children were significantly lower than those of the control group (0.244 ± 0.0179 vs. 0.261 ± 0.012 μg/l, p < 0.001, and 0.14 ± 0.13 vs. 0.24 ± 0.15 μg/l, p = 0.011, respectively). There was no significant difference between groups regarding the other serum trace element levels. In conclusion, there may be alterations in the serum levels of trace elements in obese children and these alterations may have a role in the pathogenesis of obesity.     

Serum Copper,Zinc, and Magnesium Levels in Patients with Chronic Fluorosis

Although there are many studies on effect of fluoride on trace elements in experimental animals, few studies exist on serum trace elements levels in patients with endemic fluorosis. We aimed to determine the serum levels of trace elements including serum copper (Cu), zinc (Zn), and serum levels of minerals including calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K) in patients with endemic fluorosis. The study group consisted of 30 patients with endemic fluorosis (17 females, 13 males, mean age 33.53 ± 9.85 years). An age, gender, and body mass index matched 30 healthy volunteers comprised control group (21 females, ten males with a mean age 33.93 ± 7.39 years). Urine fluoride levels of chronic fluorosis patients were significantly higher than that of control subjects as expected (1.92 ± 0.10 mg/l vs. 0.41 ± 0.09 mg/l, respectively; P < 0.001). Serum Cu levels (89.14 ± 16.77 μg/dL vs. 102.69 ± 25.04 μg/dL, respectively, P = 0.017), serum Zn levels (77.98 ± 20.58 μg/dL vs. 94.57 ± 35.87μg/dL, respectively, P = 0.032), and serum Mg levels (1.92 ± 0.18 mg/dL vs. 2.07 ± 0.31 mg/dL, respectively, p = 0.022) was significantly lower in chronic fluorosis patients than in controls. There were no statistically significant differences between the fluorosis group and control group with respect to serum levels of Na, K, Ca, and P. We concluded that chronic fluorosis is associated with reduced serum levels of Cu, Zn, and Mg.     

Serum Iron,Zinc, and Copper Concentration in Premature Graying of Hair

Premature graying of hair with unclear etiology, which is known as premature canities, is a common cause of referrals to the dermatologists. We assessed the relationship between serum iron, copper, and zinc concentrations with premature canities. This study was conducted on patients under 20 years old suffering from premature canities, having a minimum of ten gray hair fibers, and referring to university hospitals of Isfahan (Iran). The results were compared with age–sex-matched controls. Demographic data and disease characteristics were recorded for two groups. We studied serum iron, copper, and zinc concentrations of 66 patients and 66 controls using atomic absorption and Ferrozine methods. The mean age of studied cases was 17.8 ± 2.0 years, and the mean age of the onset of canities was 15.5 ± 3.2 years with no significant difference between males and females (P > 0.05). Serum copper concentration was significantly lower in patients compared with controls (90.7 ± 37.4 vs. 105.3 ± 50.2 μg/dL, P = 0.048), but serum iron concentration was significantly lower in controls compared to patients (88.8 ± 39.5 vs. 108.3 ± 48.4 μg/dL, P = 0.008). Also, there was no significant difference between patients and controls in serum zinc concentration (114.8 ± 67.8 vs. 108.2 ± 49.9 μg/dL, P = 0.285). According to these results, among copper, zinc, and iron, a low serum copper concentration may play a role in premature graying of hairs in our society. Further studies are needed to find the underlying mechanism of this relationship.     

Plasma Levels of Zinc,Copper, and Ceruloplasmin in Patients after Undergoing Laparoscopic Adjustable Gastric Banding

Laparoscopic adjustable gastric banding (LAGB) causes significant weight loss in morbidly obese adults. However, its consequences on nutritional status still remain unclear. There are a few studies determining the nutritional status after LAGB and none have focused on the serum levels of zinc (Zn), copper (Cu), and ceruloplasmin (CP). We aimed to investigate the effects of LAGB surgery on plasma Zn, Cu, and CP levels. Thirty patients with LAGB with morbid obesity were included. Blood samples were collected preooperatively and in the postoperative third month to determine plasma Zn, Cu, and CP levels. The mean preoperative and postoperative body mass indexes (BMI) were 44.9 ± 7.4 kg/m² and 44.1 ± 6.5 kg/m², respectively. The mean weight loss was 12.9 ± 3.3 kg at the postoperative third month. The postoperative Zn (500 ± 130 ng/ml), Cu (280 ± 80 ng/ml), and CP (23.9 ± 8.8 mg/dl) values were statistically significantly lower than the preooperative Zn (740 ± 230 ng/ml), Cu (370 ± 80 ng/ml) and CP (33.3 ± 15.7 mg/dl) levels (p < 0.05). Decreases in the plasma levels of Zn, Cu, and CP were seen postoperatively following LAGB surgery. The nutritional status of LAGB-applied patients should be monitored and mineral supplementation may be considered.     

Plasma Leptin,Plasma Zinc,and Plasma Copper Are Associated in Elite Female and Male Judo Athletes

The purpose of this study was to compare plasma leptin, plasma zinc, and plasma copper levels and their relationship in trained female and male judo athletes (n = 10 women; n = 8 men). Blood samples were obtained 24 h after training to measure plasma zinc, copper, and leptin levels. Subjects presented similar values to age (22 ± 2 years old), body mass index (24 ± 1 kg/m²), plasma zinc (17.2 ± 2 μmol/L), copper (12.5 ± 2 μmol/L), and leptin (5.6 ± 1.3 μg/L). However, height, total body mass, lean mass, fat mass, and sum of ten-skinfold thickness were higher in male than female. Plasma leptin was associated with sum of ten skinfolds in male (r = 0.91; p < 0.001) and female athletes (r = 0.84; p < 0.003). Plasma zinc was associated with leptin in males (r = 0.82; p < 0.05) while copper was associated with plasma leptin in females (r = 0.66; p < 0.05). Our results suggest that young judo athletes lost sex-related differences in leptin levels. Plasma zinc, plasma copper, and energy homeostasis may be involved in regulation of plasma leptin.     

The Role of Calcium,Magnesium, and Zinc in Pre-Eclampsia

Pre-eclampsia is the most common medical complication of pregnancy associated with increased maternal and infant mortality and morbidity. Its exact etiology is not known, although several evidences indicate that various elements might play an important role in pre-eclampsia. This study was carried out to analyze and to compare the concentration of calcium, magnesium, and zinc in the serum of women with pre-eclampsia and in normal pregnant women. Fifty clinically diagnosed patients with pre-eclampsia (25 with mild and 25 with severe pre-eclampsia) and 50 normal pregnant controls were enrolled in this study. The serum calcium, magnesium, and zinc levels were estimated with an atomic absorption spectrophotometer. The mean serum levels of calcium, magnesium, and zinc in normal pregnant group were 2.45 ± 0.18 mmol/L, 0.79 ± 0.13 mmol/L, and 15.64 ± 2.4 μmol/L, respectively, while in mild pre-eclamptic group, these were 2.12 ± 0.15 mmol/L, 0.67 ± 0.14 mmol/L, and 12.72 ± 1.7 μmol/L, respectively. Serum levels in severe pre-eclamptic group were 1.94 ± 0.09 mmol/L, 0.62 ± 0.11 mmol/L, and 12.04 ± 1.4 μmol/L, respectively. These results indicate that reduction in serum levels of calcium, magnesium, and zinc during pregnancy might be possible contributors in etiology of pre-eclampsia, and supplementation of these elements to diet may be of value to prevent pre-eclampsia.     

Intra-erythrocyte Magnesium Is Associated with Gamma-Glutamyl Transferase in Obese Children and Adolescents

This study aims at determining the association between markers of hepatic injury and serum, urinary, and intra-erythrocyte magnesium concentrations and dietary magnesium intake in obese children and adolescents. In a case–control study, 42 obese children and adolescents (8–18 years) and 42 sex- and puberty-matched controls were studied. Serum, urinary, and intra-erythrocyte magnesium levels, indices of insulin sensitivity, and liver enzymes were measured. Dietary magnesium intake was assessed using a food frequency questionnaire. Obese children and adolescents exhibited insulin resistance as determined by a higher fasting insulin and the HOMA-IR (p < 0.001) and lower QUICKI indices (p = 0.001); in addition these subjects had significantly higher intra-erythrocyte magnesium (IEM) concentrations, than non-obese ones (3.99 ± 1.05 vs. 3.35 ± 1.26 mg/dL of packed cell; p = 0.015). Among liver enzymes, only gamma-glutamyl transferase (GGT) was significantly higher in obese than in non-obese subjects (22.7 ± 9.4 vs. 17.1 ± 7.9 U/l; p = 0.002). A positive association was found between GGT and IEM in both groups; however in multivariate analysis, in obese subjects, only GGT (p = 0.026) and, in non-obese subjects, only age (p = 0.006) remained as significant predictors of IEM. In conclusion, increased IEM concentration was seen in insulin-resistant obese children and adolescents; furthermore, serum GGT was associated with IEM, independently of body mass index and HOMA-IR.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

Plantlet regeneration from leaf derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andr.

Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP). Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening, with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be produced within eight subcultures from callus obtained from leaf explant through the methods described here.     

Dialysability of Magnesium and Calcium from Hospital Duplicate Meals: Influence Exerted by Other Elements

Total and dialysable magnesium and calcium levels and corresponding dialysabilities were measured in duplicate meals (n = 108) during 36 consecutive days. The interaction exerted by other nutrients and energy on them was also performed. Total mean magnesium and calcium fractions of 113.9 ± 98.3 and 337.2 ± 278.9 mg/meal respectively, were found. The Mg and Ca levels supplied by meals are positively (p < 0.05) correlated with macronutrient contents (carbohydrates and proteins). The mean dialysable Mg and Ca fractions were 56.9 ± 36.3 and 127.4 ± 112.3 mg/meal (50.4 ± 13.2 and 37.8 ± 10.7% as dialysabilities, respectively). Total Mg and Ca levels are significantly correlated with corresponding element dialysabilities (p < 0.05). For both minerals, significant correlations between their total and dialysable fractions and between their dialysable level and dialysabilities were noted (p < 0.01). The mean Mg and Ca daily dietary intakes (DDI) were 341.7 ± 68.0 and 1,011.6 ± 424.4 mg/day, respectively. For Ca and Mg the existence of similarities in their behaviour in meals and absorptive processes has been found. Duplicate meals with raw vegetables are good sources of bioaccessible Mg. High Ca dialysability has been found in the analysed meals. The fish and products constitute a good source of bioaccessible Ca. Mg, Ca, zinc, and chromium levels enhanced significantly the Mg dialysability. The Ca dialysability rose significantly with dialysable Ca and chromium fractions (p < 0.05).     

Influence of Therapy with Metformin on the Concentration of Certain Divalent Cations in Patients with Non-insulin-Dependent Diabetes Mellitus

Research was performed on a group of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM), who never received antidiabetic medication before, and on a group of 17 healthy adults. The patients were administered treatment with metformin, 1,000 mg/day. Plasmatic and urinary concentration of magnesium have been measured, copper and zinc along with the concentrations of glucose, HDL, LDL, cholesterol, tryglicerides, HbA1c, and total erythrocyte magnesium, in advance and after 3 months of treatment. Data showed significant differences in the NIDDM group vs the control group: for plasma magnesium—1.95 ± 0.19 vs 2.20 ± 0.18 mg/dl, p < 0.001; urine magnesium—237.28 ± 34.51 vs 126.25 ± 38.22 mg/24 h, p < 0.001; erythrocyte magnesium—5.09 ± 0.63 vs 6.38 ± 0.75 mg/dl, p < 0.001; plasma zinc—67.56 ± 6.21 vs 98.41 ± 20.47 μg/dl, p < 0.001; urine zinc—1,347.54 ± 158.24 vs 851.65 ± 209.75 μg/24 h, p < 0.001; plasma copper—111.91 ± 20.98 vs 96.33 ± 8.56 μg/dl, p < 0.001; and urine copper—51.70 ± 23.79 vs 36.00 ± 11.70 μg/24 h, p < 0.05. Treatment with metformin for 3 months modified significant erythrocyte magnesium—5.75 ± 0.61 vs 5.09 ± 0.63 mg/dl, p < 0.001 and urine magnesium—198.27 ± 27.07 vs 237.28 ± 34.51 mg/24 h, p < 0.001, whereas it did not modify significant the plasmatic and urinary concentration of the other cations. The erythrocyte magnesium concentration was inversely correlated with HbA1c (r = −0.438, p = 0.015). The plasma level of copper was positively correlated with HbA1c (r = 0.517, p < 0.003), tryglicerides (r = 0.534, p < 0.003), and cholesterol (r = 0.440, p < 0.05), and the plasma level of zinc was inversely correlated with glycemia (r = −0.399, p = 0.029). Our data show a significant action of metformin therapy, by increasing the total intraerythrocyte magnesium concentration and decreasing the urinary magnesium elimination, positively correlated with the decrease of glycemia and HbA1c in NIDDM patients.     

Zinc in Postmortem Body Tissues and Fluids

Data on zinc concentration in the human body may be used to interpret the results obtained in cases of chronic and acute poisonings with zinc compounds, i.e., in clinical and forensic toxicology. In this paper, the concentrations of zinc in human tissues and body fluids obtained from autopsy cases concerning non-poisoned people (n = 203), aged from 14 to 80 years, between 1995 and 2008, are presented. The following values were found by the flame atomic absorption method (mean ± SD, median, range, in microgram per gram or microgram per milliliter): brain 10.3 ± 1.36, 10.2, 7.99–13.8 (n = 48); stomach 14.2 ± 3.63, 13.6, 8.00–22.5 (n = 71); intestines 15.7 ± 5.22, 15.8, 8.36–28.1 (n = 35); liver 39.6 ± 16.1, 36.6, 16.0–78.8 (n = 109); kidney 33.8 ± 10.1, 31.8, 16.4–60.9 (n = 93); lung 12.0 ± 3.88, 11.0, 6.13–18.7 (n = 26); spleen 14.7 ± 2.53, 14.6, 11.4–18.3 (n = 5); heart 26.5 ± 3.63, 26.7, 22.5–31.8 (n = 5); blood 6.81 ± 1.21, 7.00, 4.02–8.68 (n = 50); urine 0.69 ± 1.70, 0.60, 0.39–1.00 (n = 5), and bile 4.92 ± 1.64, 3.75, 3.20–7.09 (n = 9). The accuracy of the method was checked through the use of SRM Bovine Liver 1577b (certified: 127 ± 16 μg Zn/g, found: 117 ± 0.7 μg Zn/g (n = 6)).