n-3多不饱和脂肪酸和肾脏疾病

慢性肾脏疾病（CKD）已成为一个威胁公众健康的重要问题，如不及早预防与干预，将导致进行性肾脏纤维化和肾功能减退，直至进入终末期肾衰竭，而不得不依赖透析或肾移植生存。原发病的类型是决定肾功能减退速度的重要因素，除此之外，目前的研究显示许多心血管疾病的危险因素如高血压、糖尿病、吸烟和脂质代谢紊乱等亦能影响CKD的进展。鱼油中的主要成分n-3多不饱和脂肪酸（n-3PUFA）被证实具有心血管保护作用，饮食富含n-3PUFA的爱斯基摩人和日本人群中动脉粥样硬化引起的死亡比例较其他人群明显降低。CKD的病理生理学特征与动脉粥样硬化有许多相似之处，近来一些研究提示n-3PUFA对于肾脏疾病亦有益处，我们就此作一综述。     

Association of plasma n-6 and n-3 polyunsaturated fatty acids with synovitis in the knee: the MOST study

In osteoarthritis (OA) the synovium is often inflamed and inflammatory cytokines contribute to cartilage damage. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have anti-inflammatory effects whereas omega-6 polyunsaturated fatty acids (n-6 PUFAs) have, on balance, proinflammatory effects. The goal of our study was to assess the association of fasting plasma phospholipid n-6 and n-3 PUFAs with synovitis as measured by synovial thickening on contrast enhanced (CE) knee MRI and cartilage damage among subjects in the Multicenter Osteoarthritis Study (MOST). MOST is a cohort study of individuals who have or are at high risk of knee OA. An unselected subset of participants who volunteered obtained CE 1.5T MRI of one knee. Synovitis was scored in six compartments and a summary score was created. This subset also had fasting plasma, analyzed by gas chromatography for phospholipid fatty acid content, and non-CE MRI, read for cartilage morphology according to the Whole-Organ Magnetic Resonance Imaging Score (WORMS) method. The association between synovitis and cartilage morphology and plasma PUFAs was assessed using logistic regression after controlling for the effects of age, sex, and BMI. 472 out of 535 subjects with CE MRI had complete data on synovitis, cartilage morphology and plasma phospholipids. Mean age was 60 years, mean BMI 30, and 50% were women. We found an inverse relation between total n-3 PUFAs and the specific n-3, docosahexaenoic acid with patellofemoral cartilage loss, but not tibiofemoral cartilage loss or synovitis. A positive association was observed between the n-6 PUFA, arachidonic acid, and synovitis. In conclusion, systemic levels of n-3 and n-6 PUFAs which are influenced by diet, may be related to selected structural findings in knees with or at risk of OA. Future studies manipulating the systemic levels of these fatty acids may be warranted to determine the effects on structural damage in knee OA.     

Modulation of human hepatocyte acute phase protein production in vitro by n-3 and n-6 polyunsaturated fatty acids.

OBJECTIVE: The authors investigate the role of a variety of essential polyunsaturated fatty acids on the spontaneous and interleukin-6 stimulated production of acute phase proteins by isolated human hepatocytes. SUMMARY BACKGROUND DATA: The altered production of acute phase proteins by the liver is one of the principal effects of the systemic inflammatory response in human disease. It has been shown that polyunsaturated fatty acids have certain anti-inflammatory properties that potentially are mediated through altered prostaglandin or proinflammatory cytokine production. However, the effect of polyunsaturated fatty acids on the responsiveness of the human hepatocyte to proinflammatory cytokines has not been studied in detail. METHODS: Hepatocytes isolated from human livers were maintained in primary culture in the presence of a variety of bovine serum albumin-complexed fatty acids. The influence of these fatty acids on hepatocyte acute phase protein production was assessed, in the presence and absence of recombinant interleukin-6, by measurement of acute phase proteins by enzyme-linked immunosorbent assay. RESULTS: Eicosapentaenoic and gammalinolenic acid increased spontaneous production of alpha 1-antichymotrypsin and prealbumin but decreased spontaneous production of transferrin and haptoglobin from isolated human hepatocytes. Eicosapentaenoic and gammalinolenic significantly increased interleukin-6-stimulated production of C-reactive protein and alpha 1-antichymotrypsin but reversed the stimulatory effect of interleukin-6 on haptoglobin production. These fatty acids also reversed the inhibitory effect of interleukin-6 on prealbumin production. CONCLUSIONS: These results show that certain fatty acids have the potential to modulate spontaneous and cytokine-induced alterations in human hepatic acute phase protein metabolism. These data indicate the presence of complex mechanisms of regulation of human hepatic protein metabolism by fatty acids, and further study will be required to establish the nature of their influence in vivo.     

The effect of n-3 fatty acids on heart rate variability in patients treated with chronic hemodialysis.

OBJECTIVE: The aim of the present study was to address the effect of n-3 polyunsaturated fatty acids (PUFAs) on heart rate variability (HRV) in patients treated with chronic hemodialysis. DESIGN: We performed a randomized, placebo-controlled intervention trial. SETTING: The study took place at two hospital-based dialysis centers. PATIENTS: Thirty patients with documented cardiovascular disease who were treated with hemodialysis for at least 6 months were included. INTERVENTION: Treatment consisted of 1.7 g of n-3 PUFA or a control treatment (olive oil). MAIN OUTCOME MEASURE: The outcome measure was 24-hour Holter recordings with time domain HRV measurements at baseline and after 3 months of treatment. Blood samples were obtained to assess the content of n-3 PUFA in serum phospholipids before and after treatment. RESULTS: n-3 PUFA did not significantly affect time domain parameters of HRV, compared with a control group. CONCLUSION: We conclude that treatment with n-3 PUFA does not increase HRV in patients treated with chronic hemodialysis, a result that may have been compromised by a small sample size.     

Improved survival of heterotopic cardiac allografts in rats with dietary n-3 polyunsaturated fatty acids

A heterotopic cardiac transplant model, with male Fischer 344 rats as donors and Long Evans rats as recipients, was utilized to investigate the effect of dietary n-3 polyunsaturated fatty acids on acute rejection. Both donor and recipient rats were fed purified diets high in either n-3 polyunsaturated fatty acids (from concentrated n-3 ethyl esters [EE] or fish oil [FO]) or n-6 polyunsaturated fatty acids (from corn oil [CO]) for either 2-3 or 3-4 weeks before transplant. The recipient rats continued on their diets until rejection. The AIN-76A-based diets (with 30% of calories as fat) had adequate essential fatty acids and were balanced for sterols and antioxidants. Allograft survival was significantly increased by 45% when recipient rats were fed EE as compared to the control (CO diet fed to both donor and recipient), regardless of the diet fed to the donor. There was a slight but significant increase in allograft survival when only donor rats were fed the EE diet 2-3 weeks before transplant. With the FO diet (containing one third of the n-3 fatty acids in the EE diet), only the group fed FO to both donor and recipient (starting 2-3 weeks before transplant) showed a significant increase in allograft survival over the control. However, if the FO diets were fed for 3-4 weeks before transplant, increased survival was seen in groups fed FO to either the donor or recipient alone. In this case, allograft survival with FO feeding to both donor and recipient was not different from recipient treatment alone. In all the studies there was a significant and direct correlation between allograft survival and the donor heart phospholipid n-3/n-6 fatty acid ratio and the n-3 fatty acid content (at rejection). There was an indirect relationship with the n-6 fatty acid content. There was no detectable 20:3 (n-9) in the cardiac phospholipids, indicating the absence of essential fatty acid deficiency. Recipient diets were the strongest determinant of the fatty acid composition in the transplanted donor heart. The data indicate that providing dietary n-3 polyunsaturated fatty acids before and after cardiac transplant to recipient animals provides a significant protection against acute rejection.     

Reduction of delayed-type hypersensitivity by the injection of n-3 polyunsaturated fatty acids in mice.

The effects of injection of n-3 polyunsaturated fatty acids (PUFAs) on the delayed-type hypersensitivity (DTH) response was investigated in mice. Mice were immunized with sheep red blood cells (SRBCs). Six days later 50 microliters of a 20% SRBC suspension was injected into the right hind footpad of each mouse. Just before the challenge of SRBCs, various amount of a trieicosapentaenoyl-glycerol emulsion (10%) was injected through tail veins (5 mice per each dose). Then 24 hr later the dorsoventral thickness of the right hind footpad was measured and compared with that of the left hind footpad. The difference in thickness between both footpads was regarded as the DTH response. The effect of the emulsion on DTH was dose-dependent; the DTH responses (in mm) in the control group (injected with 0.5 ml of a 2.5% glycerol solution through tail veins) and EPA-injected groups (with 5 mg, 10 mg, and 20 mg) were 1.53 +/- 0.16 (mean +/- SEM), 1.09 +/- 0.14, 0.43 +/- 0.07 (P less than 0.005), and 0.36 +/- 0.13 (P less than 0.005), respectively. The DTH response was also depressed by the injection of a tridocosahexaenoyl-glycerol emulsion. Consequently, n-3 PUFA emulsions have clinical implication in DTH-related diseases such as rejection of allografts.     

Modulation of Kupffer cell membrane phospholipid function by n-3 polyunsaturated fatty acids

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been reported to improve clinical outcome in a number of inflammatory diseases including burns and sepsis. One mechanism contributing to the anti-inflammatory effect is the incorporation of n-3 PUFAs into membrane phospholipids which decreases macrophage eicosanoid production. We hypothesize that an additional mechanism for their effects is an alteration of membrane signal transduction that decreases macrophage responsiveness to inflammatory stimuli. Kupffer cells, the fixed macrophages of the liver, were obtained from rats pair fed diets for 6 weeks with 15% of calories supplied as menhaden (high n-3), corn (control), or safflower (high n-6) oils. The effects of the dietary oils on Kupffer cell membrane signal transduction and eicosanoid production were assessed by measuring inositol phospholipid (PI) metabolism, intracellular calcium responses, and prostaglandin E2 (PGE2) production to the inflammatory signals endotoxin (LPS) and platelet activating factor (PAF). The menhaden oil diet resulted in significant incorporation of n-3 PUFAs into total cellular PUFAs compared to corn and safflower oil. (total n-3 PUFAs, 28.1% menhaden vs 2.1% corn vs 1.2% safflower, P less than 0.03). This incorporation altered signal transduction of PAF as both PI turnover (65% +/- 10% of corn oil) and calcium response (0.6-fold vs 5.0-fold for corn oil) were significantly reduced in the menhaden oil group. (P less than 0.05) The menhaden oil diet also reduced significantly PGE2 production in response to PAF and LPS (corn, 348 +/- 23 pg/ml; menhaden, 48 +/- 6 pg/ml, P less than 0.01). We conclude that, in addition to modulating eicosanoid production, n-3 PUFAs can also alter macrophage membrane signal transduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum bile acids and pruritus in hemodialysis patients

BACKGROUND: Chronic renal failure (CRF) patients usually suffer from pruritus. The pathophysiology of pruritus is still incompletely understood. SUBJECTS, MATERIALS AND METHODS: In this paper we determined serum total bile acids (STBA) in hemodialysis patients with advanced CRF (ACRF) in order to obtain STBA concentration in predialysis, to assess their probable relation among patients with pruritus and in postdialysis using a polysulfone membrane for dialysis. STBA were determined in 49 ACRF patients with chronic hemodialysis and values were compared to 20 control subjects. Hemodialysis patients were divided in two groups: with and without pruritus. In all these patients, month of renal replacement therapy, diabetic patients, dose of dialysis (Kt/V), viral markers, serum creatinine, serum glucose, aspartate and alanine aminotransferase, alkaline phosphatase, hematocrits and albumin were determined. The intensity of itching among pruritic patients was measured by a score system: mild (M), moderate (MO) and severe (S). RESULTS: No significant differences were found in patients with and without pruritus in months of renal replacement therapy, duration of dialysis or dose of dialysis (Kt/V). STBA were determined in all ACRF patients in predialysis and they showed significant differences compared to controls (p < 0.05), however, no differences were observed in the results obtained when control subjects were compared to ACRF patients without pruritus. Also in predialysis, pruritic patients showed significant differences in STBA compared to patients without pruritus (p < 0.001). STBA concentration showed a significant decrease in postdialysis using a polysulfone membrane in ACRF patients with and without pruritus. Finally, correlation with STBA and itch score of pruritus was significant (p < 0.02). CONCLUSION: Hemodialysis patients with ACRF and pruritus showed an increase of STBA in predialysis and a decrease in postdialysis.     

n-3多不饱和脂肪酸对人结肠癌细胞增殖与凋亡的影响

目的探讨n-3多不饱和脂肪酸（n-3PUFA）对人结肠癌细胞HT．29的作用及其机制。方法应用MTT比色法、细胞的形态学观察（Hochest33258染色）、DNA凝胶电泳、流式细胞技术检测二十二碳六烯酸（DHA）对HT．29增殖和凋亡的影响：气相色谱分析的方法检测DHA对HT-29细胞n-3PUFA和n．6PUFA含量及n-6／n-3PUFA比例的影响。结果DHA在体外对HT．29有明显的增殖抑制作用,10、20、40和80mg儿DHA作用24h时的细胞增殖抑制率分别为16．8％、24．7％、50．0％和60．1％。40mg／LDHA作用24、48和72h的细胞增殖抑制率分别为50．0％、69．9％和77．0％：呈现明显的剂量和时间效应关系。荧光染色可观察到细胞核染色质浓集,核浓缩核碎裂．并出现典型的凋亡小体：DNA凝胶电泳呈现特征性的梯形条带（DNALadder）：流式细胞仪检测显示经DHA处理后HT-29DNA合成前期（G,期）细胞比例较对照组增加（72．1％比51．3％）,DNA合成期（S期）细胞比例明显减少（19．9％比38．9％）,细胞呈现明显的G,期阻滞;气相色谱分析显示．DHA可以降低HT-29细胞内n-6PUFA而提高n-3PUFA含量,降低n-6／n-3PUFA比率。结论n-3PUFA通过抑制细胞增殖和诱导细胞凋亡来阻遏结肠癌细胞的生长．这种作用的机制可能为降低了细胞的n-6／n-3PUFA的比例。     

Effects of n-3 polyunsaturated fatty acids on hepatic tight junction after partial hepatectomy in rats

Objective

To explore the effects of n-3 polyunsaturated fatty acid (PUFA) on the hepatic tight junction in rat liver after partial hepatectomy.

Materials and Methods

Male Sprague-Dawley rats were divided into 4 groups: sham operation, 70% hepatectomy, 70% hepatectomy with administration of 1 mL/kg n-3 PUFA, and 70% hepatectomy with administration of 2 mL/kg n-3 PUFA. Morphologic features of the hepatic tight junction were observed at transmission electron microscopy, and expression of the tight junction proteins occludin, claudin-3, and ZO-1 was studied using Western blot analysis.

Results

The hepatic tight junction structure became loosened 3 days after 70% hepatectomy. The levels of tight junction occludin decreased markedly, whereas claudin-3 and ZO-1 levels increased 2- or 3-fold over control levels. Supplementation of n-3 PUFA alleviated the changes in tight junction structure and occludin expression.

Conclusion

n-3 PUFA has protective effects on hepatic tight junction structure after 70% hepatectomy, which were attributed in part to modulation of occludin expression.     

Omega-3 polyunsaturated fatty acids and proxies of cardiovascular disease in hemodialysis: a prospective cohort study

BACKGROUND: Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have antithrombotic, lipid-lowering and antiinflammatory properties. The aim of this study was to verify if dietary supplementation with omega-3 PUFAs is able to induce changes of blood pressure, nutritional and coagulative profile, inflammation and blood cell counts in patients on hemodialysis (HD). METHODS: We designed a 12-month, prospective, single-blind, sequential intervention, cohort study. All of the HD patients undergoing HD in our unit were eligible for the study. Patients on HD for at least 6 months with an autologous vascular access were enrolled. No thresholds for blood pressure or lab parameters were considered. Patients taking nonsteroidal antiinflammatory drugs, steroids or statins or those with catheters, grafts, liver diseases, malignancies, malnutrition or sepsis were excluded. After the baseline evaluations the patients underwent 3 consecutive 4-month study periods taking the following supplements: A (olive oil: 2 g/day), B (omega-3 PUFA: 2 g/day), C (olive oil: 2 g/day). RESULTS: Twenty-four patients met the inclusion criteria. All patients completed the follow-up. Fibrinogen, hemoglobin, platelet, red and white blood cell counts, parathormone (PTH), partial thromboplastin time (PTT), serum total cholesterol, triglycerides, apolipoprotein A and B, C-reactive protein (CRP) and albumin levels did not change significantly during the study. On the contrary, systolic (mean +/- SD) (A: 131 +/- 17.8 mm Hg; B: 122 +/- 12.8 mm Hg; C: 129 +/- 13.2 mm Hg), diastolic (A: 83 +/- 16.3 mm Hg; B: 72 +/- 14.8 mm Hg; C: 79 +/- 6.5 mm Hg) and mean blood pressure (A: 99 +/- 16.8 mm Hg; B: 88 +/- 14.1 mm Hg; C: 96 +/- 8.7 mm Hg) were significantly lower at the end of study period B (repeated measures ANOVA and Tukey's post hoc test: p<0.05). CONCLUSIONS: In our experience, blood pressure was the only parameter influenced by omega-3 PUFA supplementation in patients on long-term HD.     

Influence of topical administration of n-3 and n-6 essential and n-9 nonessential fatty acids on the healing of cutaneous wounds.

Injury triggers a series of physiological events at the wound site. These include an inflammatory response that is established shortly after the injury, which is then followed by an intense formation of tissue over a period of days. Poly- and monounsaturated fatty acids exert major functions on the inflammatory responses, either in the form of phospholipids anchored in the cell membrane or as soluble lipoic mediators. We present evidence that linolenic (n-3), linoleic (n-6), and oleic (n-9) fatty acids can modulate the closure of surgically induced skin wounds. We found that n-9 fatty acids induced faster wound closure when compared to n-3, n-6, and control. In addition, n-9 fatty acids strongly inhibited the production of nitric oxide at the wound site. A mild improvement on wound closure was observed in the n-6 fatty acid-treated animals concurrent with a peak in nitric oxide production at 48 hours postsurgery. N-3 fatty acid treatment significantly delayed wound closure. Furthermore, we showed that n-3 fatty acid induced a peak in nitric oxide at 3 hours postsurgery and an intense deposition of extracellular matrix after 5 days of treatment. Thus, our results suggest a relevant role and potential therapeutic implication for fatty acids on skin wound healing.     

Prostate tissue and leukocyte levels of n-3 polyunsaturated fatty acids in men with benign prostate hyperplasia or prostate cancer

OBJECTIVE: To compare the levels of n-3 polyunsaturated fatty acids (PUFAs) in leukocytes and prostate tissue in men with prostate cancer or benign prostatic hyperplasia (BPH), as dietary intake of n-3 PUFAs has been linked to the risk of prostate cancer; the prostate-specific antigen (PSA) level was also compared to prostate tissue levels of n-3 PUFAs. PATIENTS AND METHODS: Prostate tissue was obtained and leukocytes isolated from 20 men with prostate cancer and 35 with BPH. The n-3 PUFAs alpha-linolenic acid (ALA), eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) were measured in prostate tissue and in peripheral blood leukocytes using gas chromatography. PSA levels were measured in all of the men. RESULTS: There was a strong positive correlation between EPA and DHA in leukocytes and in prostate tissue (EPA: r = 0.80, DHA: r = 0.53, both P < 0.001) in all the men, whereas there was no association between the content of ALA in leukocytes and in prostate tissue (r = -0.15). Men with BPH had similar levels of ALA in leukocytes and in prostate tissue, but men with prostate cancer had more ALA in prostate tissue than in leukocytes. The PSA level was significantly positively correlated with ALA level in prostate tissue (r = 0.42, P < 0.01) but there was no significant correlation between PSA level and EPA and DHA levels. There were no significant correlations between PSA level and n-3 PUFA levels in leukocytes. CONCLUSION: Dietary intake of the marine n-3 PUFAs reflected in EPA and DHA levels in leukocytes are also reflected in EPA and DHA levels in prostate tissue in men with and without prostate cancer. However, there is a discrepancy between the levels of ALA in leukocytes and in prostate tissue, with higher levels in men with prostate cancer. This is in accordance with the strong positive association between PSA and ALA levels in prostate tissue. This study therefore does not support the hypothesis that intake of marine n-3 PUFAs might protect against prostate cancer, but lends support to the deleterious role of ALA in the development of prostate cancer.     

The effect of n-3 fatty acids on C-reactive protein levels in patients with chronic renal failure.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of mortality in patients with chronic renal failure (CRF). C-reactive protein (CRP), a strong independent risk marker of CVD, is elevated in a large proportion of patients with CRF. The long-chain n-3 polyunsaturated fatty acids (PUFA) have cardioprotective effects, which may be partly attributed to their anti-inflammatory properties. OBJECTIVE: The study objective was to investigate the effect of n-3 PUFA on serum levels of CRP in patients with CRF. DESIGN: We performed a randomized, double-blind, placebo-controlled study. SETTING: The study took place at an outpatients clinic at the Department of Nephrology, Aalborg Hospital, Denmark. PATIENTS: The study comprised 46 patients (30 men and 16 women; mean age 59 +/- 11 years) with a serum creatinine level in the range of 150 to 400 micromol/L. INTERVENTION: The patients were randomly assigned to daily supplementation with 2.4 g n-3 PUFA or identical capsules containing 2.4 g of olive oil (control) for 8 weeks. MAIN OUTCOME MEASURE: CRP was measured with a high-sensitivity C-reactive protein (hs-CRP) assay and the content of n-3 PUFA in granulocyte membranes before and after supplementation. RESULTS: The n-3 PUFA concentration increased in granulocytes after the n-3 PUFA supplements but was unaltered by the control oil. A nonsignificant reduction in hs-CRP was observed in the n-3 PUFA group after supplementation (2.46 vs. 1.47 mg/L; P = .06), and hs-CRP was unaltered by the control oil (3.27 vs. 3.14 mg/L; P = .12). There was no difference in median hs-CRP change in the two groups. CONCLUSION: A trend was seen toward a reduction in hs-CRP in the n-3 PUFA group, but there was no significant difference in hs-CRP levels when both groups were compared.     

Short-term administration of omega-3 fatty acids in hemodialysis patients with balanced lipid metabolism.

BACKGROUND: At present, it is discussed whether omega-3 fatty acids show anti-inflammatory, antithrombogenic, and antiatherosclerotic effects, also in patients with chronic renal failure. METHODS: In this prospective study, 11 hemodialysis (HD) patients, ages 59 +/- 17 years, who had balanced lipid metabolism and had been on HD for 53 +/- 47 months, were treated with a moderate dose of omega-3 fatty acids (1.2 g/day combined with 11.2 g/day pectin) for 12 weeks. Serum concentrations of c-reactive protein, homocysteine (Hcy), lipids, complement factors, blood gas analyses, 24-hour blood pressure, heart rate variability, electrocardiography, shunt blood flow, and recirculation, as well as peripheral oxygen saturation at the hand and foot, were measured at the start (t0w), and after 12 weeks (t12w) of therapy. Results Several assessed cardiovascular risk factors were significantly influenced. Levels of very-low-density lipoproteins (t0w, 77 +/- 26; t12w, 63 +/- 32 mg/dL; P <.05) and triglycerides (t0w, 261 +/- 157; t12w, 228 +/- 131 mg/dL; P =.068) were decreased. However, Hcy concentrations increased from 35.5 +/- 32.5 to 43.5 +/- 36.7 micromol/L ( P <.01) after 12 weeks. Anti-inflammatory and investigated clinical parameters did not significantly change during the study period. CONCLUSION: Limited positive effects on metabolic parameters were evaluated by short-term administration of omega-3 fatty acids in HD patients. Based on previous studies and on suspicion of atherosclerotic disorder in examined HD patients, we suppose that only high doses of omega-3 fatty acids given for a longer time influence inflammation and atherosclerosis.     

Immunosuppression with polyunsaturated fatty acids in renal transplantation.

A double-blind controlled trial has been undertaken to assess the value of a preparation containing polyunsaturated fatty acids (PUFA) in human cadaveric renal transplantation. Eighty-nine patients were studied and followed for 6 months after transplantation. Forty-four took the PUFA preparation and 45 the placebo (oleic acid). Other immunosuppression was standardised. Functional graft survival was significantly better in the PUFA group than in those taking the placebo during the first 3 to 4 months post-transplant. At 6 months, however, although the difference between the groups persisted, it was no longer statistically significant. Complications were equally distributed between the groups.