Safety evaluation of a medium- and long-chain triacylglycerol oil produced from medium-chain triacylglycerols and edible vegetable oil.

To reduce the incorporation of dietary lipids into adipose tissue, modified fats and oils have been developed, such as medium-chain triacylglycerols (MCT). Typical dietary lipids from vegetable oils, termed long-chain triacylglycerols (LCT), are degraded by salivary, intestinal and pancreatic lipases into two fatty acids and a monoacyl glycerol; whereas, MCT are degraded by the same enzymes into three fatty acids and the simple glycerol backbone. Medium-chain fatty acids (MCFA) are readily absorbed from the small intestine directly into the bloodstream and transported to the liver for hepatic metabolism, while long-chain fatty acids (LCFA) are incorporated into chylomicrons and enter the lymphatic system. MCFA are readily broken down to carbon dioxide and two-carbon fragments, while LCFA are re-esterified to triacylglycerols and either metabolized for energy or stored in adipose tissue. Therefore, consumption of MCT decreases the incorporation of fatty acids into adipose tissue. However, MCT have technological disadvantages precluding their use in many food applications. A possible resolution is the manufacture and use of a triacylglycerol containing both LCT and MCT, termed medium- and long-chain triacylglycerol (MLCT). This manuscript describes studies performed for the safety evaluation of a MLCT oil enzymatically produced from MCT and edible vegetable oil (containing LCT), by a transesterification process. The approximate fatty acid composition of this MLCT consists of caprylic acid (9.7%), capric acid (3.3%), palmitic acid (3.8%), stearic acid (1.7%), oleic acid (51.2%), linoleic acid (18.4%), linolenic acid (9.0%), and other fatty acids (2.9%). The approximate percentages of long (L) and medium (M) fatty acids in the triacylglyerols are as follows: L, L, L (55.1%), L, L, M (35.2%), L, M, M (9.1%), and M, M, M (0.6%). The studies included: (1) acute study in rats (LD50>5000 mg/kg); (2) 6 week repeat-dose safety study via dietary administration to rats (NOAEL of 3500 mg/kg/day), (3) in vitro genotoxicity studies using Salmonella typhimurium and Escherichia coli (negative at 5000 mg/plate), and (4) a four-week, placebo-controlled, double blind, human clinical trial utilizing 20 test subjects (no effects at 42 g MLCT/day). These data are corroborated by other studies published in the peer-reviewed literature on analogous MLCTs.

A potent antiviral effect on hepatitis C viral dynamics in serum and peripheral blood mononuclear cells during combination therapy with high-dose daily interferon alfa plus ribavirin and intravenous twice-daily treatment with interferon beta.

Hepatitis C virus (HCV) is known to infect and replicate within peripheral blood mononuclear cells (PBMC), thereby enabling the direct evaluation of antiviral mechanisms by analyzing HCV dynamics in PBMC. To address potential molecular differences associated with distinct antiviral regimens, we studied HCV dynamics in both serum and PBMC in 44 patients with HCV genotype 1b and high viral load who were randomly assigned to the following 4 different treatment groups: 1) combination therapy with 6 MU daily of interferon alfa 2b (IFN-alpha2b) plus 800 mg of ribavirin; 2) monotherapy with 6 MU daily of IFN-alpha2b; 3) monotherapy with twice-daily intravenous administration with 3MU of IFN-beta; and 4) monotherapy with daily intravenous administration with 6 MU of IFN-beta. HCV-RNA levels were measured serially using highly sensitive real-time detection polymerase chain reaction (PCR). HCV dynamics in both the serum and PBMC showed a "biphasic" pattern. The exponential decay slopes of the second phase were significantly higher in the combination or twice-daily dosing regimen groups compared with groups 2 or 4 (0.10 +/- 0.08 vs. 0.02 +/- 0.09 or 0.16 +/- 0.09 vs. 0.02 +/- 0.04 day(-1); P <.05 or P <.0005, respectively). Moreover, the viral half-lives in the second phase were significantly shorter in these groups (73.2 +/- 42.5 vs. 240.1 +/- 120.7 or 56.0 +/- 44.6 vs. 361.6 +/- 293.5 hours; P <.05 or P <.05, respectively). Additionally, the slope of HCV decline in PBMC tended to be higher in the combination regimens, as compared with monotherapy. Taken together, our data on HCV dynamics provide molecular insight into utilization of combination or twice-daily dosing regimens to increase rates of sustained viral eradication of HCV.

Risk factors for distant recurrence of hepatocellular carcinoma in the liver after complete coagulation by microwave or radiofrequency ablation.

BACKGROUND: In patients with hepatocellular carcinoma (HCC), recurrences in the distant liver often are observed after curative treatment. Microwave coagulation therapy (MCT) and radiofrequency ablation (RFA) have been developed as less invasive alternatives than surgical resection for small HCCs. In the current study, risk factors for distant recurrence of HCC were analyzed in patients in whom complete coagulation was achieved. METHODS: Ninety-two patients with HCCs < 3 cm in greatest dimension were treated by MCT or RFA percutaneously or laparoscopically. Eighty-four patients in whom complete coagulation was achieved without recurrence in the same subsegment as the primary nodule were included in this study. Distant recurrences were observed in 22 patients. Fifteen possible risk factors for a distant recurrence were analyzed. RESULTS: When comparing the patients with a recurrence of HCC nodules in the remnant liver to those without recurrence, the authors observed a statistically significant difference only in serum alpha-fetoprotein. The distant recurrence-free survival was analyzed by the Kaplan-Meier method. A statistically significant difference was observed in hepatitis C virus (HCV) infection as an etiopathic agent of underlying liver diseases (P < 0.005) and in the number of the primary HCC nodules (P < 0.05, log-rank test). A multivariate stepwise Cox hazard model revealed that HCV infection and the number of primary HCC nodules were statistically independent risk factors. CONCLUSIONS: Patients who had more than two HCC nodules accompanied by HCV infection had a high incidence of recurrence of HCC in the remnant liver, even when coagulation by microwave or ablation by radiofrequency was complete.

[Cranial fasciitis of a neonatal case with massive intra- and extracranial extension].

We report a neonatal male case of cranial fasciitis in childhood. He was born with a large skull tumor in his left fronto-parietal region. The day after his birth, he was admitted to our hospital. On inspection, the mass was firm, non mobile, and had two humped peaks. The size of the mass was 7 x 5 x 4 cm, and it was located on the left coronal suture and fontanelle. The overlying skin was normal. Skull X-rays revealed osteolytic defect and linear bone remnant skirted the outer rim of the tumor. CT scan showed a slightly high-density large frontal mass with a low density area in the center of it and marked enhancement. MR images revealed a large mass with massive intra- and extracranial extension. Cerebral angiography showed the remarkable vascularization of the tumor. Preoperatively, the branches of the external carotid artery were partly embolized. On surgery, the profusely bleeding, elastic hard, yellowish-white mass arising from the dura was partially removed with the surrounding bone. A small mass remained subdurally. The boundary of the mass and the dura was not well demarcated. After the operation, no neurological deficits were seen. 16 months after the surgery, MRI revealed the unfolded brain with no evidence of tumor. Histological examination showed characteristic proliferation of spindle-shaped fibroblasts embedded in a collagenous stroma. Inflammatory lymphocytes and acidophils were also noted. Electron micrograph revealed a tumor cell rich in rough endoplasmic reticulum and nuclei with numerous indentations. Immunostaining confirmed that these cells were positive for vimentin but negative for smooth muscle actin, GFAP, S-100 protein, and desmin. No recurrence of this tumor has been detected at four years of follow-up.

Determination of amphetamine and methamphetamine in human hair by headspace solid-phase microextraction and gas chromatography with nitrogen-phosphorus detection.

A simple and rapid method for the determination of amphetamine (AP) and methamphetamine (MA) in human hair was developed by headspace solid-phase microextraction (SPME) and gas chromatography with nitrogen-phosphorus detection (GC-NPD). The hair (1 mg) was dissolved in 0.2 ml of a 5 M sodium hydroxide solution in a tightly sealed vial by shaking at 75 degrees C for about 5 min. In order to adsorb AP and MA on the SPME fiber, 100 microm of polydimethylsiloxane fiber was exposed to the headspace of the vial, and the vial was heated at 55 degrees C for 20 min. Then the fiber was removed from the vial and inserted into the injection port of the GC-NPD system using a CBJ-17 capillary column. The compounds adsorbed on the fiber were analyzed by exposing the fiber at 220 degrees C for 30 s in the GC injection port. By using this method, AP and MA in human hair could be analyzed simply and rapidly without any interference from coexisting substances. The percentages of AP and MA extracted from human hair by the SPME method were 48 and 62%, respectively, and relative standard deviations were below 10% (n=5). The calibration curves for AP and MA were linear in the ranges of 0.4-15 and 4-160 ng/mg hair, respectively. The detection limits of AP and MA at a signal-to-noise ratio of three were 0.1 and 0.4 ng/mg hair, respectively. This method could be applied to the analysis of an abuser's hair sample.

Flow cytometric method to detect lymphocyte transformation in drug-allergic hepatic injury.

Flow cytometric methods for the analysis of incorporated bromodeoxyuridine are extremely rapid and simple. We investigated whether these methods were useful for detecting drug-allergic hepatic injury in 18 patients with drug-allergic hepatic injury, 18 healthy controls, and 9 nonallergic patients receiving drugs. Peripheral blood mononuclear cells were stimulated with drug solutions. Incorporation of bromodeoxyuridine was detected after labeling with FITC, and S-phase cells were counted by flow cytometry. Percentages of S-phase cells in drug-stimulated culture minus those in spontaneous cultures were less than 1% in both healthy controls and nonallergic patients receiving drugs. Taking 1% as the upper limit, 13 patients (72%) were judged as positive. After the in vitro addition of interleukin-2, two patients among five who had been judged as negative were judged as positive. Lymphocyte transformation test by flow cytometry may be useful in the diagnosis of drug-allergic hepatic injury.

Inhibition by piroxicam of oxidative DNA damage, liver cirrhosis and development of enzyme-altered nodules caused by a choline-deficient, L-amino acid-defined diet in rats.

Previously, we have reported that aspirin, a cyclooxygenase (COX) inhibitor, can prevent the fibrosis, cirrhosis and generation of oxidative DNA damage, and the associated development of glutathione-S-transferase placental form (GST-P)-positive preneoplastic liver nodules, caused by a choline-deficient, L-amino acid-defined (CDAA) diet in rats. In the present study, in order to elucidate the role of COX pathway in liver lesion-induction by a CDAA diet, the modulatory effects of other distinct chemical classes of COX inhibitors were examined. A long-acting example, piroxicam (PIRO) (at doses of 0.01, 0.02, 0.04 and 0.06%) and the short-acting ibuprofen (IBU) (at doses of 0.02, 0.04 and 0.06%) and indomethacin (IND) (at doses of 0.005 and 0.008%) were administered in the CDAA diet to male F344 rats, and animals were killed after 12 and 30 weeks. In another experiment, IND was given in drinking water at doses of 0.001, 0.002 and 0.004%. None of the inhibitors affected the development of fatty liver caused by a CDAA diet, but PIRO at doses higher than 0.04%, strongly inhibited the development of GST-P-positive and neoplastic nodules as well as fibrosis, cirrhosis and formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts. IBU at the highest dose also exhibited similar but much less pronounced inhibitory effects. With IND, there was only a tendency for inhibition with no clear dose-dependence. The results together with our previous findings, indicate that relatively strong COX inhibitors, acting irreversibly like aspirin or for extended periods like PIRO, can prevent the endogenous hepatocarcinogenesis associated with a CDAA diet, although not the development of a fatty liver, suggesting that an augmented COX pathway might play key roles in the causation of liver lesions in this model.

Hemolysis, elevated liver enzymes, and low platelets syndrome associated with primary anti-phospholipid antibody syndrome.

A 28-year-old woman with a history of a spontaneous abortion developed thrombocytopenia, Coombs-negative hemolytic anemia, and liver dysfunction at the sixteenth week of pregnancy. These findings were compatible with hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome (hemolysis, elevated liver enzymes, and low platelet counts). Moreover, serum anti-phospholipid antibodies were positive, suggesting the association of anti-phospholipid antibody syndrome. An artificial abortion, anti-coagulation therapy, and plasma exchange were performed concomitantly with corticosteroid therapy. She responded to the therapy, a remission was obtained. Anti-phospholipid antibodies may play a role in the pathogenesis of HELLP syndrome.

Effect of alcohol drinking on gene expression of hepatic O6-methylguanine DNA methyltransferase in chronic liver diseases.

O6-methylguanine DNA methyltransferase (MGMT) is a repair protein that transfers methyl groups from O6-methylguanine to a cysteine acceptor in its own molecule, and restores DNA to its undamaged state. If left unrepaired, O6-methylguanine can pair with either a thymine or a cytosine, causing a C-G to T-A transition, which is considered to be one of the molecular mechanisms of both mutagenesis and carcinogenesis. The expression of MGMT mRNA in liver tissue was quantitatively assessed by the competitive reverse transcription-polymerase chain reaction method in patients with chronic liver diseases with or without alcohol drinking. MGMT mRNA expression was 1.4 +/- 0.9 pg/micrograms RNA in control livers. Its expression in chronic hepatitis was 3.8 +/- 0.7 in alcoholics and 2.7 +/- 0.8 in nonalcoholics, which were not statistically different. MGMT mRNA expression in liver cirrhosis was significantly low, compared with that in chronic hepatitis, and 0.8 +/- 0.3 in alcoholics and 0.5 +/- 0.1 in nonalcoholics, which also were not significantly different. The present study shows that MGMT mRNA was not decreased in patients with chronic liver diseases with alcohol drinking, compared with those without alcohol drinking.

Hepatic iron contents and response to interferon-alpha in patients with chronic hepatitis C. Relationship to genotypes of hepatitis C virus.

Recent reports have shown that response to interferon treatment is influenced by hepatic iron contents in patients with chronic hepatitis C. In those reports, however, hepatitis C virus (HCV) genotypes and serum HCV-RNA levels were not examined. The aim of the present study was to investigate whether hepatic iron contents influence the response to interferon in patients with chronic hepatitis C and whether HCV genotypes and serum HCV-RNA levels play a role in this relationship. Among 65 patients with chronic hepatitis C, hepatic iron contents were significantly high in patients with a history of excess drinking of alcohol (more than 80 g/day) compared to those without, and significantly low in female patients before menopause. Having excluded these patients, hepatic iron contents were significantly higher in patients with genotype 1b infection than those with genotype 2a and 2b infection. There was no significant correlation between hepatic iron contents and plasma HCV-RNA levels. Among the patients with genotype 1b infection, hepatic iron contents were significantly lower in the responders to interferon than those in the nonresponders (429 +/- 100 vs 875 +/- 110 micrograms/g liver, P < 0.05). From these results, it is concluded that response to interferon is mainly influenced by HCV genotypes, while hepatic iron contents may play an important role in response to interferon in patients with genotype 1b infection.

Improvement of peripheral blood lymphocyte subsets in primary biliary cirrhosis after ursodeoxycholic acid therapy.

Immunological abnormalities frequently observed in patients with primary biliary cirrhosis are considered to be related to the pathogenesis of this disease. We performed a prospective trial to evaluate whether immune mechanisms play a role in the effectiveness of ursodeoxycholic acid (UDCA) therapy. Fifteen female patients with primary biliary cirrhosis were followed for 1 year and were then treated with UDCA (600 mg/day) for another year. Laboratory tests, including peripheral blood lymphocyte subsets assessed by dual colour fluorescence analysis using monoclonal antibodies against respective T cell markers, were evaluated at the beginning of the study, at the start of therapy and at the end of therapy. In primary biliary cirrhosis, the proportion of cytotoxic T cells, suppressor inducer T cells and alpha beta-receptor bearing T cells were significantly lower than in healthy controls. No significant changes were observed in the proportions during the year before the therapy. These reductions, however, recovered to normal ranges after 1 year of UDCA therapy. These changes were associated with an improvement in the serum levels of aspartate aminotransferase, alkaline phosphatase, gamma-globulin and IgM. The close correlation between the improvement in the imbalance of lymphocyte subsets after the therapy and the clinical status suggests that an immunological process may play a role in the effectiveness of therapy in primary biliary cirrhosis.

Effects of cell proliferation and cell death (apoptosis and necrosis) on the early stages of rat hepatocarcinogenesis.

An experiment was performed to investigate whether, during regression of the liver hyperplasia induced by a direct mitogen, apoptosis differentially affects replicated and non-replicated hepatocytes. After a single dose of the direct mitogen lead nitrate (LN), male Wistar rats were given repeated injections of tritiated thymidine, and were killed either 3 days (time of maximal hepatic DNA increase) or 15 days (complete regression of the hyperplasia) after mitogen treatment. Determination of liver DNA radioactivities and labelling indices (LIs) at the two time points revealed an approximately 40% loss in total liver DNA radioactivity, a 20% decrease in the specific activity of DNA, and a 20% reduction in the cell LI. Three days after LN administration 64% of the apoptotic bodies contained thymidine grains in their nuclear fragments. The results indicated that apoptosis affects both hepatocytes that replicated, and those that did not replicate, the former being slightly more sensitive. A second experiment was then performed to investigate whether and to what extent different types of cell death (apoptosis versus necrosis) influence the growth of hepatocytes initiated by a chemical carcinogen. Male Wistar rats were given a single dose of diethylnitrosamine, and 2 weeks thereafter either a single dose of LN, or a necrogenic dose of carbon tetrachloride (CCl4). Bromodeoxyuridine was next infused for 5 days, and some of the animals were killed at this time point, and others after an additional 3 weeks. Administration of CCl4 resulted in an increase in both the average size and the percentage area occupied by placental glutathione S-transferase-positive lesions. In contrast, administration of lead nitrate resulted in a strong reduction (50%) in the number of positive lesions with no remarkable change in the percentage area occupied by them. These differential effects occurred even though comparable LIs were observed in rats treated with the two agents. The results suggest that lead nitrate leads to a loss of initiated hepatocytes, due to the apoptosis that occurs during regression of the LN-induced hyperplasia.

Inhibition by acetylsalicylic acid, a cyclo-oxygenase inhibitor, and p-bromophenacylbromide, a phospholipase A2 inhibitor, of both cirrhosis and enzyme-altered nodules caused by a choline-deficient, L-amino acid-defined diet in rats.

Effects of inhibitors of arachidonic acid (AA) metabolism on the development of fatty liver, cirrhosis, glutathione-S-transferase placental form (GST-P)-positive nodules and the generation of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances (TBARS), caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male Fischer 344 rats by feeding CDAA diets supplemented with the inhibitors for 12 and 30 weeks. Acetylsalicylic acid (ASA) (at doses of 0.1 and 0.2%) and p-bromophenacylbromide (BPB) (0.1 and 0.2%) were used as inhibitors of, respectively, cyclo-oxygenase and phospholipase A2, and quercetin (QU) (0.75 and 1.5%) and nordihydroguaiaretic acid (NDGA) (0.1 and 0.2%) as inhibitors of lipoxygenase. None of the inhibitors affected the development of fatty liver caused by the CDAA diet. ASA at a doe of 0.2% almost completely prevented the appearance of cirrhosis, GST-P-positive nodules, 8-OHdG and TBARS in seven out of 11 (63.7%) rats. BPB at a dose of 0.2% also exerted inhibitory effects on all of these lesions but to a lesser extent than ASA. QU and NDGA exerted inhibitory effects limited to the GST-P-positive nodule case. The results indicate that a perturbed AA metabolism, particularly of the cyclo-oxygenase pathway, derived secondarily from depletion of labile methyl groups or phosphatidylcholine, might play key roles in the cirrhosis, hepatocarcinogenesis and oxidative stress caused by a CDAA diet. The results also indicated a possible involvement of the lipoxygenase pathway in hepatocarcinogenic processes.

Gene expressions of c-met and hepatocyte growth factor in chronic liver disease and hepatocellular carcinoma.

BACKGROUND/AIMS: The roles of c-met proto-oncogene and hepatocyte growth factor in human livers have not been shown. METHODS: Gene expressions of both c-met and hepatocyte growth factor were quantified in livers with chronic active hepatitis and in cirrhotic livers with hepatocellular carcinoma as well as in normal controls, using competitive reverse transcription polymerase chain reaction. RESULTS: C-met expression was significantly increased in chronic active hepatitis compared with control livers, and c-met expression in chronic active hepatitis correlated with serum alanine aminotransferase levels. Hepatocyte growth factor expression was increased in some patients with chronic active hepatitis compared with controls, and there was a significant correlation between c-met expression and hepatocyte growth factor expression. On the other hand, in hepatocellular carcinoma tissues, c-met expression was increased in some cases, while that in the surrounding non-carcinomatous tissues was similar to normal controls. Hepatocyte growth factor expression was not detected in the hepatocellular carcinoma tissues and was low in the surrounding non-carcinomatous tissues. CONCLUSIONS: These findings suggest that hepatocyte growth factor may be involved in the regeneration of hepatocytes via paracrine mechanism in chronic active hepatitis, while in regulation of c-met expression in hepatocellular carcinoma tissues may be independent of hepatocyte growth factor stimulation.

Effects of additional administration of colchicine in ursodeoxycholic acid-treated patients with primary biliary cirrhosis: a prospective randomized study.

BACKGROUND/AIMS: Although ursodeoxycholic acid is effective for the treatment of primary biliary cirrhosis, some patients do not respond to this treatment. The objective of the present study was to investigate the effects of additional administration of colchicine in ursodeoxycholic acid-treated patients with primary biliary cirrhosis. METHODS: Twenty-two patients with primary biliary cirrhosis treated with ursodeoxycholic acid (600 mg/day) for 30 months were randomly assigned to two groups: group 1, colchicine (1 mg/day) and ursodeoxycholic acid (n = 10); group 2, ursodeoxycholic acid alone (n = 12). RESULTS: In group 1, there were significant decreases in mean serum levels of alkaline phosphatase, total bilirubin, gamma-glutamyltranspeptidase, alanine aminotransferase, aspartate aminotransferase, and IgM, and these changes were more remarkable in those who responded poorly to ursodeoxycholic acid. In contrast, there were no significant changes in those values in group 2. CONCLUSIONS: Additional administration of colchicine to ursodeoxycholic acid may be beneficial for patients with primary biliary cirrhosis, especially those who respond poorly to ursodeoxycholic acid. It is necessary, however, to further confirm the efficacy of colchicine by examining histological changes and following the patients for longer periods.

Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue.

Using two different commercially available extraction kits, genomic/nuclear DNA could be recovered from rat liver samples as small as 10 mg. Background 8-hydroxydeoxyguanosine levels in such DNA were low and stable at 0.26-0.30 +/- 0.01-0.03/10(5) guanine residues. The minimum tissue wet weight required for the accurate 8-hydroxydeoxyguanosine analysis was 25 mg. The present results indicate that routine and reliable assessment of the involvement of oxidative DNA damage in the development of various diseases, including cancer, is feasible using a variety of tissue sources.

Initiation of hepatocarcinogenesis by endogenously formed N-nitrosobis(2-hydroxypropyl)amine, N-nitrosodiethanolamine and N-nitroso-2,6-dimethylmorpholine in rats.

Initiation activities of endogenously formed N-nitrosobis(2-hydroxypropyl)amine (NBHPA), N-nitrosodiethanolamine (NDELA) and N-nitroso-2,6-dimethylmorpholine (NDMM) were investigated in a modified short-term assay for rat hepatocarcinogenesis. Male Wistar rats were fed 1% bis(2-hydroxypropyl)amine, 0.5% diethanolamine or 0.25% 2,6-dimethylmorpholine in the diet plus 0.3% sodium nitrite in the drinking water. Two weeks after starting the experimental regimen they underwent 2/3 partial hepatectomy and were then maintained on the respective diets for a further week. Following a 2 week recovery period on basal diet rats were subjected to a resistant hepatocyte regimen consisting of 0.02% 2-acetylaminofluorene in the diet for 2 weeks and 1 mg carbon tetrachloride/kg body wt by gavage at the midpoint. Initiation activity was assayed by measuring hepatic foci positive for gamma-glutamyltranspeptidase. Numbers of such foci per cm2 were significantly increased in the groups given the secondary amines together with nitrite compared with values for groups given each precursor or nitrite alone. Further, the numbers of lesions were essentially similar to those found in rats given carcinogenic doses of NBHPA, NDELA and NDMM. The results clearly of demonstrate hepatocyte initiation activities of endogenously formed carcinogens, presumably NBHPA, NDELA and NDMM.