Conjugated Linoleic Acid Administration Research Articles

Cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid gastric administration during the pregestational and gestational periods does not influence the follicular endowment of the progeny.

Fetal programming suggests that maternal stimulation and nutrition during the period of fetal development can program the progeny. Conjugated linoleic acid (CLA), an isomer of linoleic acid, has been characterized in several aspects, but few studies have been performed on its involvement in reproduction and fetal programming. The aim of this study was to evaluate the F1, F2 and F3 progeny of female mice supplemented with CLA during the pregestational and gestational periods with respect to biometric and reproductive parameters, as well as ovarian morphophysiology. The F1 progeny of mothers supplemented with CLA exhibited stable weight gain, while the F2 progeny showed no effects (P=0.0187 and P=0.0245, respectively). A reduction in Lee's Index was observed in both generations at the second post-weaning evaluation week in the animals treated with CLA (P=0.0100 and P=0.0078, respectively). The F2 generation showed an increase in the anogenital index in both sexes of the animals treated with CLA (P= 0.0114 and P<0.0001, female and male respectively). CLA administration to mothers did not affect any of the following in their progeny: ovarian follicle mobilization (P>0.05), follicle number (P>0.05) and the integrated density of the lipid content of oocytes included in antral follicles (P>0.05). This study evaluated the use of CLA in mothers and found that it did not affect the progeny regarding murine reproductive performance, suggesting that this supplement can be used safely.

Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid supplementation on maternal reproductive parameters.

Conjugated linoleic acid (CLA) is a mixture of positional isomers of linoleic acid found in ruminant products and meat. The diet supplementing with CLA is an emerging area, requiring studies to elucidate its effects on animals and human reproduction, as well as its side effects. Therefore, the aim of this study was to evaluate the effects of CLA gastric administration, during the pregestational and gestational period in biometric and reproductive parameters, as well as in ovarian morphophysiology. Animals were distributed in three groups: (1) control (n = 10); (2) fish oil (n = 10); and (3) CLA (n = 10), that daily received, by gavage, phosphate-buffered saline, fish oil and CLA, respectively, carried out over 50 days (before mating, mating and pregnancy). There was an increment in the nasoanal distance and Lee index of the CLA and fish oil-treated groups during the first weeks (P > 0.05). CLA administration did not affect the ovarian follicle mobilization (P > 0.05), the number of follicles (P > 0.05) and the integrated density of lipid content of oocytes included in antral follicles (P > 0.05). There was no effect of CLA administration on the litter weight (P > 0.05; F2 and F3), however, an increment (P < 0.05) in the number of pups per litter (F2) was observed. Overall, this study demonstrated the absence of side effects of the CLA gastric administration on mice reproductive performance and suggests that this treatment would transgenerationally enhance fertility in this species.

Conjugated linoleic acid attenuates neuropathic pain induced by sciatic nerve in mice

Purpose: Conjugated linoleic acid (CLA) has been suggested to be necessary for human health, but there is limited research regarding its effect on neuropathic pain (NP). Here, we aim to investigate the potential effect of CLA administration on NP development and nerve recovery.&#x0D; Methods: Forty mice were divided into four equal groups randomly. The mice in control group underwent a sham operation to achieve a unilateral sciatic nerve cut. Other groups were subjected to partial sciatic nerve ligation (PSNL) surgery followed by 4 weeks of CLA treatment. Behavioral tests were performed shortly before mice were sacrificed. Blood, sciatic nerve and spinal cord tissues were collected after sacrifice. Electron microscopy was performed to determine myelin thickness and calculate myelin thickness/axon diameter ratio.&#x0D; Results: Mice that received daily oral CLA treatment for 4 weeks after PSNL surgery showed less mechanical and thermal allodynia than mice in PSNL surgery alone group. Behavioral tests showed that CLA treatment was associated with marked increases in both nerve conduction velocity (NCV) and force of gastrocnemius contraction. In addition, CLA reduced the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), sciatic myeloperoxidase (MPO) activity, and activating transcription factor-3 (ATF-3) expression. CLA also restored mitochondrial manganese superoxide dismutase (MnSOD) activity which was decreased in the sciatic nerves and spinal cords of the PSNL surgery group. Regeneration of myelins and axons in nerve fibers in CLA group was faster and more complete than that in the vehicle group.&#x0D; Conclusion: The current study demonstrates that CLA effectively attenuates NP and significantly inhibits neuro-inflammation and oxidative stress. This treatment improves sciatic nerve form and function after injury, suggesting that it can attenuate NP.

Effect of Conjugated Linoleic Acid Supplementation on Serum Leptin Concentration: A Systematic Review and Meta-Analysis.

There are controversies regarding the effect of conjugated linoleic acid (CLA) on serum leptin. To conduct a meta-analysis of randomized controlled trials (RCTs) to assess the effect of CLA on serum leptin concentrations. Databases such as Ovid, PubMed/Medline, SCOPUS, Google Scholar, and ISI databases up to January 2017 were searched. The searches included RCTs conducted among human adults, and studies on the effect of conjugated linoleic acid on serum leptin concentrations as outcome variables. The mean difference and standard deviation of leptin changes in the intervention and control groups were used as effect size measures for the meta-analysis. Eleven trials with thirteen effect sizes were pooled in this meta-analysis. CLA supplementations could not reduce serum leptin levels significantly (-0.12 (ng/ml); 95% CI: -1.29, 1.05; P=0.837). However, the impact of CLA supplementation differed by sex and BMI status. Compared with the control group, CLA administration reduced serum leptin levels significantly in trials conducted among male (- 0.86 (ng/ml); 95% CI: -1.11, -0.62; P<0.0001) or overweight individuals (-1.37 (ng /ml); 95% CI: -2.55, -0.20; P=0.022) and lasted for less than 8 weeks (-0.90 (ng/ml); 95% CI: -1.64, -0.17; P=0.0.016). CLA supplementation might be able to decrease circulating leptin levels in studies with duration of less than 8 weeks especially among male and overweight subjects. Additional RCTs that are well controlled for energy intakes may be necessary to explain the cause of short- and long-term effects of conjugated linoleic acid. The protocol was registered with PROSPERO (No. CRD42017059165).

The effects of conjugated linoleic acids on breast cancer: A systematic review.

Recently prevention strategies for breast cancer are focused on lifestyle modification such as diet. Some dietary factors such as Conjugated linoleic acid (CLA) can lower the risk of breast cancer, metastasis and some factors concerning this malignancy. Many studies have been established in this field, but their results are inconsistent. Therefore, we evaluated this association based on systematic review among published scientific literature. We performed an electronic search using PubMed, Cochrane, Scopus, Google Scholar and Persian database (Iran Medex, magiran) to identify relevant studies. We summarized the findings of 8 papers in this review. Although, three cohort studies were not overall identified a protective effect of CLA dietary intake or CLA content in breast tissue on breast cancer incidence, metastasis and death, one of them showed an inverse association after adjusting for age. Also, among case-control studies a weak inverse association between breast cancer risk and CLA dietary intake and serum levels among post-menopausal women was reported. Besides, a clinical trial showed that some indicator of breast tumor decreased after CLA administration among women with breast adenocarcinoma. Lacking published evidence suggested inconsistent results. So, further well-designed studies are required, particularly in considering the main breast cancer risk factors.

A proof of principle clinical trial to determine whether conjugated linoleic acid modulates the lipogenic pathway in human breast cancer tissue

Conjugated linoleic acid (CLA) is widely used as a "nutraceutical" for weight loss. CLA has anticancer effects in preclinical models, and we demonstrated in vitro that this can be attributed to the suppression of fatty acid (FA) synthesis. We tested the hypothesis that administration of CLA to breast cancer patients would inhibit expression of markers related to FA synthesis in tumor tissue, and that this would suppress tumor proliferation. Women with Stage I-III breast cancer were enrolled into an open label study and treated with CLA (1:1 mix of 9c,11t- and 10t,12c-CLA isomers, 7.5 g/d) for ≥ 10 days before surgery. Fasting plasma CLA concentrations measured pre- and post-CLA administration, and pre/post CLA tumor samples were examined by immunohistochemistry for Spot 14 (S14), a regulator of FA synthesis, FA synthase (FASN), an enzyme of FA synthesis, and lipoprotein lipase (LPL), the enzyme that allows FA uptake. Tumors were also analyzed for expression of Ki-67 and cleaved caspase 3. 24 women completed study treatment, and 23 tumors were evaluable for the primary endpoint. The median duration of CLA therapy was 12 days, and no significant toxicity was observed. S14 expression scores decreased (p = 0.003) after CLA administration. No significant change in FASN or LPL expression was observed. Ki-67 scores declined (p = 0.029), while cleaved caspase 3 staining was unaffected. Decrements in S14 or Ki-67 did not correlate with fasting plasma CLA concentrations at surgery. Breast tumor tissue expression of S14, but not FASN or LPL, was decreased after a short course of treatment with 7.5 g/day CLA. This was accompanied by reductions in the proliferation index. CLA consumption was well-tolerated and safe at this dose for up to 20 days. Overall, CLA may be a prototype compound to target fatty acid synthesis in breast cancers with a "lipogenic phenotype".

Conjugated linoleic acid modulates immune responses in patients with mild to moderately active Crohn's disease

Conjugated linoleic acid (CLA) has demonstrated efficacy as an immune modulator and anti-inflammatory compound in mouse and pig models of colitis. We investigated the immunoregulatory efficacy of CLA in patients with mild to moderate Crohn's disease (CD). Thirteen patients with mild to moderately active CD were enrolled in an open-label study of CLA (6 g/d orally) for 12 weeks. Peripheral blood was collected at baseline, 6 and 12 weeks after treatment initiation for isolation of peripheral blood mononuclear cells for functional analyses of lymphoproliferation and cytokine production. Disease activity was calculated using the CD activity index (CDAI) and quality of life was assessed using the Inflammatory Bowel Disease Questionnaire (IBDQ). CLA significantly suppressed the ability of peripheral blood CD4+ and CD8+ T cell subsets to produce IFN-γ, TNF-α and IL-17 and lymphoproliferation at week 12. There was a statistically significant drop in CDAI from 245 to 187 (P = 0.013) and increase in IBDQ from 141 to 165 (P = 0.017) on week 12. Oral CLA administration was well tolerated and suppressed the ability of peripheral blood T cells to produce pro-inflammatory cytokines, decreased disease activity and increased the quality of life of patients with CD.

Conjugated linoleic acid (CLA) during last week of gestation and lactation alters colostrum and milk fat composition and performance of reproductive sows

The objectives of this experiment were to study the effect of dietary supplement of a commercial source of CLA, supplied either in the gestation/lactation period or during the post-weaning period, on sow and piglet performance and on the transfer of CLA into colostrum and milk. The experiment was designed to assess both the effect of dietary CLA supplementation to the sow in lactation and on the piglets in the nursery. There were four treatments for nursery piglets arranged factorially, two dietary treatments for origin sows (0g/kg vs 10g/kg CLA) and two treatments for weaned piglets (0g/kg vs 10g/kg CLA). Immediately after parturition, 10mL of colostrum was collected from the functional glands of each sow. On day 23 after farrowing, milk samples were also collected. Lower backfat thickness was observed after 28 days of lactation in all groups, and the loss was higher for the control than CLA group (P<0.004). No effect of dietary treatment was observed for colostrum crude fat content, but a lower concentration of crude fat was observed in milk from sows fed the diet containing CLA (P<0.01). Feeding sows a CLA (10g/kg) enriched diet during gestation and lactation increased the concentration of CLA and altered the concentration of fatty acids in colostrum and milk. Dietary CLA produced higher saturated fatty acids (SFA) and lower monounsaturated fatty acids (MUFA) concentration in all cases, but no alteration in total polyunsaturated fatty acids (PUFA) concentration. Dietary CLA had a positive influence on the C17:0 (P=0.007), C18:0, SFA, c9, t11 and t10, c12 proportions (P<0.001) and a negative effect on C16:1n-7 (P<0.001), MUFA (P<0.05), MUFA/SFA (P<0.01), C16:1n-7/C16:0 and C18:1n-9/C18:0 (P<0.001) ratios in the colostrum. Dietary CLA had a positive influence on the C14:0, C15:1, C16:0, C18:0, c9, t11, t10, c12, C20:0, C20:1, C20:5n-3 and SFA proportions (P<0.001) and a negative effect on C16:1n-9, C16:1n-7, C17:1, C18:1n-9, C20:4n-6, MUFA, MUFA/SFA, C16:1n-7/C16:0 and C18:1n-9/C18:0 (P<0.001) in milk. CLA administration to sows reduced backfat thickness loss during the lactation period and led to higher piglet weight at weaning. No effect of sow or piglet diet was observed on the main productive characteristics.

Effect of dietary CLA administration on fatty acid composition and lipogenic and lipolytic enzyme activities in suckling and weaned piglets

The objective of this study was to determine the effects of conjugated linoleic acid (CLA) administration during the lactation and post-weaning period or in the post-weaning period exclusively on fatty acid (FA) composition and enzymatic regulation in suckling and in 63-d-old piglets. During the nursery phase, 18 piglets individually penned were used and three dietary treatments ( n = 6) were applied: (1) piglets that proceeded from control sows fed a control diet (0 g CLA/kg) (CT-CT), (2) piglets from control sows fed a diet containing 10 g CLA/kg (CT-CLA) and (3) piglets from CLA fed sows fed diets containing 10 g CLA/kg (CLA-CLA). Suckling piglets (28 d of age) from sows fed the CLA enriched diet showed higher concentration of saturated FA (SFA), lower concentration of total monounsaturated FA (MUFA) and lower MUFA/SFA, C16:1 n-7/C16:0 and C18:1 n-9/C18:0 ratios in subcutaneous backfat than those from sows fed the control diet ( P<0.05). No CLA isomers were detected at weaning in the subcutaneous backfat of piglets from sows fed the control diet, but 0.44 g/100 g total FA (TFA) c9, t11-CLA and 0.12 g/100 g TFA t10, c12-CLA were observed in piglets from CLA fed sows. Also, increased c9, t11-CLA, t10, c12-CLA and SFA proportions ( P<0.05) and decreased MUFA and MUFA/SFA and C16:1 n-7/C16:0 ratios were found in the liver of piglets from CLA fed sows ( P<0.05). In 63-d-old piglets, malic enzyme activity in subcutaneous fat was lower in groups in which CLA was fed to sows and piglets or only to piglets than in the group that did not receive any CLA supplementation ( P<0.05). No effect of dietary CLA on heart L-3-hydroxyacyl-CoA dehydrogenase and glucose-6-phosphate dehydrogenase and malic enzyme activity in liver was observed. The concentration of c9, t11-CLA and t10, c12-CLA at weaning was negligible in piglets from sows fed the control diet, but after being fed for 35 d the diet enriched with CLA, the concentration was similar to that of piglets fed milk and diet containing CLA. Little alteration of enzyme regulation induced by CLA feeding was found in piglets either at 28 or 63 d of age, except for malic enzyme activity, which was lower in 63-d-old piglets fed the CLA enriched diet after weaning.

Interaction between dietary conjugated linoleic acid and calcium supplementation affecting bone and fat mass

Dietary conjugated linoleic acid (CLA) has shown wide biologically beneficial effects, such as anticancer, antiatherosclerotic, antidiabetic, immunomodulating, and antiobesity effects. However, the effects of CLA on total body ash, reflective of bone mineral content, have not been consistent. We hypothesized that the inconsistency of the CLA effect on ash may be linked to interaction between CLA and dietary calcium levels. Thus, we investigated the effects of CLA on body ash in conjunction with various calcium levels. Male ICR mice were fed three different levels of calcium (0.01, 0.5, and 1%) with or without 0.5% CLA for 4weeks for Experiment 1 and separate CLA isomers at 0.22% level with 1% calcium in Experiment 2. CLA feeding reduced body fat regardless of dietary calcium level, whereas CLA supplementation increased body ash compared to control only in animals fed the 1% calcium. In Experiment 2 it was confirmed that this observation was associated with the trans-10, cis-12 CLA isomer, but not with the cis-9, trans-11 isomer. CLA administration with 1% dietary calcium significantly improved total ash percent (%) in femurs, confirming that CLA has the potential to be used to improve bone mass.

Conjugated Linoleic Acid (CLA) inhibits new vessel growth in the mammalian brain

Conjugated Linoleic Acid (CLA) is fatty acid found endogenously in food sources that prevents new tumor development and reduces the growth of existing tumors in laboratory animals. CLA exerts its anti-carcinogenic effect by reducing VEGF and bFGF serum levels and by blocking flk-1 receptors, thereby inhibiting vascular growth critical to tumor growth and survival. Although the ability of CLA to inhibit angiogenesis in the peripheral nervous system is well characterized, it remains unknown whether CLA also affects vascular morphology in the central nervous system. Therefore, in the present study, exercising and sedentary animals received either standard rat chow or a specially formulated diet consisting of 0.5% CLA for 24 days. The brains were then examined to determine the extent of vascular growth in the cerebellum, a region known to exhibit robust exercise-induced angiogenesis. Our results indicate that CLA administration significantly reduces angiogenesis in the cerebellum. This study is the first to demonstrate the anti-angiogenic effect of CLA in the brain, and suggests that CLA be explored as a therapeutic treatment for cancer and tumors in the brain.

The t10,c12 isomer of conjugated linoleic acid stimulates mammary tumorigenesis in transgenic mice over-expressing erbB2 in the mammary epithelium

Conjugated linoleic acid (CLA), a family of isomers of octadecadienoic acid, inhibits rat mammary carcinogenesis, angiogenesis, and lung metastasis from a transplantable mammary tumor. c9,t11-CLA, the predominant isomer in dairy products, and t10,c12-CLA, a component of CLA supplements, are equally effective. The objective of the current studies was to test the efficacy of these two CLA isomers in a clinically relevant breast cancer model. Transgenic mice over-expressing erbB2 in the mammary epithelium were fed control or 0.5% CLA-supplemented diets continuously from weaning. Unexpectedly, t10,c12-CLA stimulated lobular hyperplasia of the mammary epithelium and accelerated mammary tumor development, decreasing median tumor latency to 168 days of age compared with 256 and 270 days in the c9,t11-CLA and control groups, respectively. Metastasis was also increased by t10,c12-CLA, with percentage of tumor-bearing mice with lung metastasis 73, 14 and 31% in the t10,c12-CLA, c9,t11-CLA and control groups, respectively. A second study, in which CLA administration was initiated after puberty, confirmed the stimulatory effect of t10,c12-CLA on mammary tumor development and metastasis. Additionally, t10,c12-CLA, but not c9,t11-CLA, increased the size of the liver, heart, spleen and mammary lymph node. The effects of t10,c12-CLA were not specific to erbB2 transgenic mice, as t10,c12-CLA supplementation increased proliferation in the mammary epithelium of both wild-type FVB and FVB/erbB2 mice. Moreover, the number of terminal end buds, the mammary epithelial structures most sensitive to a carcinogenic insult, was increased 30-fold in FVB wild-type mice fed t10,c12-CLA. These data suggest that it would be prudent to avoid CLA supplements containing the t10,c12-CLA isomer. However, even though c9,t11-CLA was not efficacious in the erbB2 model, its ability to inhibit mammary tumor development in rat models suggests that it may have activity for prevention of some types of breast cancer.

Conjugated linoleic acid enhances glutathione synthesis and attenuates pathological signs in MRL/MpJ-Faslpr mice

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs.

Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier

Conjugated linoleic acid (CLA) is able to reduce adiposity by affecting lipid metabolism. In particular, CLA administration to mice reduces body fat mass with a concomitant lipid accumulation in the liver. We investigated the effects of CLA on the activity of the mitochondrial citrate carrier (CIC), which is implicated in hepatic lipogenesis. The transport activity of the CIC, measured both in intact mitochondria and in the proteoliposomes, progressively increased with the duration of CLA feeding. An increase in the CIC activity of approximately 1.7-fold was found in 16 week CLA-treated mice with respect to control animals. A kinetic analysis showed a 1.6-fold increase in the V(max) of citrate transport but no change in the K(m) value. Western blot experiments revealed an increase of approximately 1.7-fold in the expression of CIC after CLA treatment. A strict correlation between the increase in CIC activity and the stimulation of the cytosolic lipogenic enzymes was also found. These data indicate that the CIC may play a role in the onset of hepatic steatosis in CLA-fed mice by supplying the carbon source for de novo fatty acid synthesis.

Synergistic effects of conjugated linoleic acid and chromium picolinate improve vascular function and renal pathophysiology in the insulin‐resistant JCR:LA‐cp rat

Conjugated linoleic acid (CLA) is a natural constituent of dairy products, specific isomers of which have recently been found to have insulin sensitizing and possible antiobesity actions. Chromium is a micronutrient which, as the picolinate (CrP), has been shown to increase insulin sensitivity in animal models, including the JCR:LA-cp rat. We tested the hypothesis that these agents may have beneficial synergistic effects on the micro- and macrovasculopathy associated with hyperinsulinaemia and early type 2 diabetes. Insulin-resistant cp/cp rats of the JCR:LA-cp strain were treated with mixed isomers of CLA (1.5% w/w in the chow) and/or CrP at 80 microg/kg/day (expressed as Cr) from 4 weeks of age to 12 weeks of age. Plasma insulin, lipid and adiponectin levels, aortic vascular function, renal function and glomerular sclerosis were assessed. CLA administration reduced food intake, body weight and fasting insulin in JCR:LA-cp rats. Plasma adiponectin levels were significantly elevated in rats treated with both CLA and CrP. Aortic hypercontractility was reduced and the relaxant response to the nitric oxide-releasing agent acetylcholine (Ach) was increased in CrP-treated rats. Striking reductions were also observed in the level of urinary albumin and the severity of glomerular sclerosis in rats treated specifically with CLA. CLA and CrP have beneficial effects ameliorating several of the pathophysiologic features of an insulin-resistant rat model. These supplements may be useful adjuncts in the management of patients with the metabolic syndrome and warrant further study.

Effect of soy protein isolate and conjugated linoleic acid on the growth of Dunning R-3327-AT-1 rat prostate tumors.

Epidemiologic and animal model studies suggest that consumption of soy isoflavones may be associated with reduced risk of prostate cancer (PC). In addition, animal model studies suggest that conjugated linoleic acid (CLA), a natural positional isomer of linoleic acid, inhibits tumor growth in various models, including models of PC. Based on the above-mentioned data, the objective of the present study was to test the hypothesis that supplementation of the diet with combinations of isoflavone-rich soy protein isolate and CLA would act to inhibit the growth of androgen-independent R-3327-AT-1 rat prostate tumor cells inoculated ectopically into male Copenhagen rats. The results of this study indicate that neither an isoflavone-rich soy protein isolate (SPI), nor CLA inhibit the in vivo growth and development of prostate tumor cells when administered in the diet either singly or in combination. Moreover, at the highest concentrations SPI and CLA (i.e., 20% SPI, 1% CLA), there was a statistically significant increase in tumors volume over controls. Administration of SPI at 10% in the diet also enhanced tumor growth, whereas at 5%, SPI exerted no measurable effect. CLA administration alone had no observable effects on AT-1 tumor growth. These results, in an established rat model, suggest caution in using isoflavone-rich SPI in human studies involving advanced hormone-refractory prostate cancer until further investigation of these effects are completed.