The Adipose Organ Is a Unitary Structure in Mice and Humans.

Obesity is the fifth leading cause of death worldwide. In mice and humans with obesity, the adipose organ undergoes remarkable morpho-functional alterations. The comprehension of the adipose organ function and organization is of paramount importance to understand its pathology and formulate future therapeutic strategies. In the present study, we performed anatomical dissections, magnetic resonance imaging, computed axial tomography and histological and immunohistochemical assessments of humans and mouse adipose tissues. We demonstrate that most of the two types of adipose tissues (white, WAT and brown, BAT) form a large unitary structure fulfilling all the requirements necessary to be considered as a true organ in both species. A detailed analysis of the gross anatomy of mouse adipose organs in different pathophysiological conditions (normal, cold, pregnancy, obesity) shows that the organ consists of a unitary structure composed of different tissues: WAT, BAT, and glands (pregnancy). Data from autoptic dissection of 8 cadavers, 2 females and 6 males (Age: 37.5 ± 9.7, BMI: 23 ± 2.7 kg/m2) and from detailed digital dissection of 4 digitalized cadavers, 2 females and 2 males (Age: 39 ± 14.2 years, BMI: 22.8 ± 4.3 kg/m2) confirmed the mixed (WAT and BAT) composition and the unitary structure of the adipose organ also in humans. Considering the remarkable endocrine roles of WAT and BAT, the definition of the endocrine adipose organ would be even more appropriate in mice and humans.

Clinic blood pressure, ambulatory blood pressure and cardiac structural alterations in nonagenarians and in centenarians.

scant information is available on the alterations in cardiac structure and function characterizing very elderly people as well as on their relationships to clinic and ambulatory blood pressure (BP) values. In 106 subjects aged 95.3 ± 3.7 years (mean ± standard deviation, 89 nonagenarians and 17 centenarians) in good clinical conditions and living in the municipal house in Milan, we measured, along with standard clinical and laboratory variables, clinic BP, 24-h ambulatory BP and echocardiographic parameters. Forty-five of the recruited subjects were normotensive individuals, whereas 61 were treated hypertensive patients. Subjects with an age greater than 90 years showed clinic systolic (SBP) and diastolic BP (DBP) both within the normal range, with values that for clinic SBP were slightly lower than the corresponding 24-h SBP (120.8 ± 15.9 vs 128.0 ± 16.3 mmHg) and for DBP slightly higher (69.7 ± 8.8 vs 64.9 ± 8.0 mmHg). Daytime average mean BP was slightly lower than night-time average mean BP, indicating the attenuation of the BP reduction during night-time. Left ventricular mass index (LVMI) was increased and significantly related to both 24-h and clinic BP values (r = 0.24, p < 0.04 and r = 0.20, p < 0.05). Thus in nonagenarians and centenarians, abnormalities in left ventricular pattern are of frequent detection and may be related both to the ageing process and to BP load.

Synthesis and biological activity of Akt/PI3K inhibitors.

Phosphatidylinositol 3-kinase (PI3K) and serine/threonine protein kinase B (PKB or Akt) pathways regulate important cellular processes and are related to a number of human pathologies, such as cancer. The development of kinase inhibitors, with particular attention to small molecule analogues of natural phosphoinositides for pathway interruption and therapeutic applications will be reviewed.

Randomized clinical trial of pylorus-preserving duodenopancreatectomy versus classical Whipple resection-long term results.

BACKGROUND: It is not known whether pylorus-preserving duodenopancreatectomy is as effective as the classical Whipple procedure in the resection of pancreatic and periampullary tumours. A prospective randomized trial was undertaken to compare the results of the two procedures. METHODS: Clinical data, histological findings, short-term results, survival and quality of life of all patients having surgery for suspected pancreatic or periampullary cancer between June 1996 and September 2001 were analysed. RESULTS: Two hundred and fourteen patients were randomized to undergo either a standard or a pylorus-preserving Whipple resection. After exclusion of 84 patients on the basis of intraoperative findings, 130 patients (66 standard Whipple operation and 64 pylorus-preserving resection) were entered into the trial. Of these, 110 patients with proven adenocarcinoma (57 standard Whipple and 53 pylorus-preserving resection) were analysed for long-term survival and quality of life. There was no difference in perioperative morbidity. Long-term survival, quality of life and weight gain were identical after a median follow-up of 63.1 (range 4-93) months. At 6 months, capacity to work was better after the pylorus-preserving procedure (77 versus 56 per cent; P = 0.019). CONCLUSION: Both procedures were equally effective for the treatment of pancreatic and periampullary cancer. Pylorus-preserving Whipple resection offers some minor advantages in the early postoperative period, but not in the long term.

Oxygen extraction in pigs subjected to low-dose infusion of endotoxin after major abdominal surgery.

BACKGROUND: Sepsis may impair O(2) extraction due to blood flow redistribution or decreased utilization of the available oxygen. METHODS: We assessed the effect of endotoxemia on systemic and regional O(2) extraction and lactate handling in pigs, randomized to receive either endotoxin (0.4 microg kg(-1) h(-1); n = 10) or saline infusion (controls; n = 9) for 12 h. RESULTS: High baseline regional and systemic O(2) extraction in the endotoxin group (median 56%, range 45-77%) and in the controls (67%, 49-72%) was maintained until the end of the experiment (endotoxin group: 60%, 50-71%; controls: 60%, 50-74%) despite hypotension and a decrease in stroke volume in endotoxic animals. Hepatic lactate exchange decreased during endotoxemia from 14 micromol kg(-1) min(-1) (range 10-28 micromol kg(-1) min(-1)) to 10 (range 3-15) micromol kg(-1) min(-1); P < 0.01), but remained stable in the controls, with 13 micromol min(-1) (4-18 micromol min(-1)) at baseline and 7 micromol min(-1) (3-17 micromol min(-1)) after 12 h of saline infusion. CONCLUSIONS: The high and sustained oxygen consumption and oxygen extraction in this endotoxemic model speak against any major impairment of hepatosplanchnic or systemic oxygen extraction and oxidative metabolism. The reduced hepatic lactate exchange despite an unchanged hepatic lactate influx suggests altered metabolic activities independent of oxygen consumption.

[Compared progression of visual-evoked potentials in preterm and term newborns]. / Evolution comparée des potentiels évoqués visuels chez des enfants nés à terme et prématurément.

PURPOSE: To compare the development of the visual pathway in healthy preterm and full-term newborns using flash and patterned (both transient and steady-state) visual-evoked potentials (fVEP, pVEP-t, pVEP-ss). PATIENTS AND METHODS: Twenty-nine preterm newborns (28-35 weeks gestational age) were evaluated at four different times: at 3 months postnatal and corrected age, at 8 months postnatal and corrected age. They were matched with 92 term newborns tested at 3 and 8 months of life. RESULTS: The maximal perceived spatial frequencies in the groups were: 2 C/d at 3 months postnatal age, >2 C/d at 3 months corrected age and at 3 months at term, between 3 and 5C/d, >=5C/d at 8 months corrected age and at 8 months of life, respectively. The latencies of the P1 and P100 waves for the flash and the transient stimulation were, respectively, 192 ms and 207.9 ms at 3 months postnatal age, 144 ms and 137.7 ms at 3 months corrected age, 126.3 ms and 124.1 ms at 8 months postnatal age, 112.7 ms and 112.5 ms at 8 months corrected age, 137.3 and 110.1 ms at 3 months of life; and 122.3 and 100.5 ms at 8 months of life. DISCUSSION: In the preterm infants of 3 gestational months, the latencies of the fVEP are comparable to those of the 3-month-old full-term infants, although the pVEP-t latencies are still delayed at 8 months. In the fVEP; however, we noted a shorter latency in the preterm infants at 8 months corrected age compared to those of the full-term infants. This tendency inversion was also evidenced at the steady-state stimulation. CONCLUSIONS: As evidenced by our results, at 8 months of corrected age the preterm infants recovered the fVEP and pVEP-ss, but not the pVEP-t. Different results obtained with different visual-evoked stimulations suggest that similar neural pools are developed in not completely equal amounts of time.

Curative resection is the single most important factor determining outcome in patients with pancreatic adenocarcinoma.

BACKGROUND: Mortality rates associated with pancreatic resection for cancer have steadily decreased with time, but improvements in long-term survival are less clear. This prospective study evaluated risk factors for survival after resection for pancreatic adenocarcinoma. METHODS: Data from 366 consecutive patients recorded prospectively between November 1993 and September 2001 were analysed using univariate and multivariate models. RESULTS: Fifty-eight patients (15.8 per cent) underwent surgical exploration only, 97 patients (26.5 per cent) underwent palliative bypass surgery and 211 patients (57.7 per cent) resection for pancreatic adenocarcinoma. Stage I disease was present in 9.0 per cent, stage II in 18.0 per cent, stage III in 68.7 per cent and stage IV in 4.3 per cent of patients who underwent resection. Resection was curative (R0) in 75.8 per cent of patients. Procedures included pylorus-preserving Whipple resection (41.2 per cent), classical Whipple resection (37.0 per cent), left pancreatic resection (13.7 per cent) and total pancreatectomy (8.1 per cent). The in-hospital mortality and cumulative morbidity rates were 2.8 and 44.1 per cent respectively. The overall actuarial 5-year survival rate was 19.8 per cent after resection. Survival was better after curative resection (R0) (24.2 per cent) and in lymph-node negative patients (31.6 per cent). A Cox proportional hazards survival analysis indicated that curative resection was the most powerful independent predictor of long-term survival. CONCLUSION: Resection for pancreatic adenocarcinoma can be performed safely. The overall survival rate is determined by the radicality of resection. Patients deemed fit for surgery who have no radiological signs of distant metastasis should undergo surgical exploration. Resection should follow if there is a reasonable likelihood that an R0 resection can be obtained.

1 alpha,25-Dihydroxycholecalciferol reduces rejection and improves survival in rat liver allografts.

Vitamin D(3) affects the immuno response and improves experimental autoimmune diseases. We investigated the effect of 1,25-dihydroxycholecalciferol (1,25[OH](2)D(3)) Rocaltrol as a single immunosuppressive agent and in combination with low-dose cyclosporin A (CsA) in vascularized liver allografts in rats in a high-responder strain combination (ACI-->Lewis). Recipients were placed on a low-calcium diet 7 days before transplantation and were treated with 0.1 or 1 microg/kg/d 1,25(OH)(2)D(3) intraperitoneally beginning 3 days before transplantation. Treatment combining 1,25(OH)(2)D(3) with CsA (2 mg/kg/d) was also tested. Graft function and survival, histologic rejection, and concentrations of interleukin (IL)-2, -4, -10, and -12 in serum and in grafts were measured. 1,25(OH)(2)D(3) increased allograft survival in a dose-dependent manner when compared with controls (P <.05 for both groups). Serum bilirubin, aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities were significantly lower in 1,25(OH)(2)D(3)-treated animals. Vitamin D reduced the concentration of IL-2 and IL-12 in serum and in grafts, and increased IL-4 and IL-10 in the grafts. The rejection activity index 10 days after transplantation was significantly lower in low- and high-dose 1,25(OH)(2)D(3)-treated rats compared with vehicle-treated controls (P <.0001 for both groups). The combination of either low-dose or high-dose vitamin D(3) and CsA prolonged graft survival when compared with low-dose CsA only (P <.05 for both groups). After 3 weeks, hypercalcemia developed in high-dose 1,25(OH)(2)D(3)-treated rats. It is concluded that 1,25(OH)(2)D(3) prolongs survival of liver allografts in rats by decreasing the severity of acute rejection. Analogues of vitamin D with fewer hypercalcemic effects may have potential as immunosuppressive drugs in liver transplantation.

Intrathecal levels of complement-derived soluble membrane attack complex (sC5b-9) correlate with blood-brain barrier dysfunction in patients with traumatic brain injury.

It has become evident in recent years that intracranial inflammation after traumatic brain injury (TBI) is, at least in part, mediated by activation of the complement system. However, most conclusions have been drawn from experimental studies, and the intrathecal activation of the complement cascade after TBI has not yet been demonstrated in humans. In the present study, we analyzed the levels of the soluble terminal complement complex sC5b-9 by ELISA in ventricular cerebrospinal fluid (CSF) of patients with severe TBI (n = 11) for up to 10 days after trauma. The mean sC5b-9 levels in CSF were significantly elevated in 10 of 11 TBI patients compared to control CSF from subjects without trauma or inflammatory neurological disease (n = 12; p < 0.001). In some patients, the maximal sC5b-9 concentrations were up to 1,800-fold higher than in control CSF. The analysis of the extent of posttraumatic blood-brain barrier (BBB) dysfunction, as determined by CSF/serum albumin quotient (Q(A)), revealed that patients with a moderate to severe BBB impairment (mean Q(A) > 0.01) had significantly higher intrathecal sC5b-9 levels as compared to patients with normal BBB function (mean Q(A) < 0.007; p < 0.0001). In addition, a significant correlation between the individual daily Q(A) values and the corresponding sC5b-9 CSF levels was detected in 8 of 11 patients (r = 0.72-0.998; p < 0.05). These data demonstrate for the first time that terminal pathway complement activation occurs after head injury and suggest a possible pathophysiological role of complement with regard to posttraumatic BBB dysfunction.

Pylorus-preserving total pancreatectomy. Early and late results.

BACKGROUND/AIMS: Preservation of the pylorus is an accepted alternative procedure to the classical Whipple operation for pancreatic head resection but data describing its value for total pancreatectomy are sparse. METHODS: A prospective analysis of 22 total pancreatectomies performed in a consecutive series of 436 pancreatic resections from 1.11.93 to 1.5.99. RESULTS: 11 patients underwent total pancreatectomy with preservation of the pylorus. Histopathological examination revealed pancreatic adenocarcinoma in 16 cases and duodenal adenocarcinoma in 1 patient, 5 patients had other types of pancreatic neoplasm. In-hospital mortality was 4.5% (n = 1), cumulative morbidity was 59% and reoperations were performed in 9.1% of cases (n = 2). Median follow-up was 37 months (range 5-66). 62% of patients (n = 13) developed tumor recurrence and 13 patients died during the follow-up period with 10 deaths being cancer related. There was no difference concerning postoperative and follow-up morbidity of survival between patients undergoing pylorus-preserving total pancreatectomy or pancreatectomy with gastrectomy. However, postoperative body weight was increased 3, 6, 9 and 12 months following preservation of the pylorus. CONCLUSION: Total pancreatectomy with preservation of the pylorus is a feasible type of resection for all types of pancreatic or ampullary tumors, which shows a similar morbidity and long-term survival but improved nutritional recovery compared with standard total pancreatectomy.

Enzymatic properties of human hemalbumin.

The binding of hemin to the primary site of human serum albumin (HSA) has been reinvestigated using UV-Vis, CD and NMR techniques. The major fraction of bound hemin contains a five-coordinated high-spin iron(III) center, but a minor fraction of the metal appears to be in a six-coordinated, low-spin state, where a 'distal' residue, possibly a second histidine residue, completes the coordination sphere. The reduced, iron(II) form of the adduct contains six-coordinated low-spin heme. The distal residue hinders the access to the iron(III) center of hemin-HSA to small anionic ligands like azide and cyanide and destabilizes the binding of neutral diatomics like dioxygen and carbon monoxide to the iron(II) form. In spite of these limitations, the hemin-HSA complex promotes hydrogen peroxide activation processes that bear the characteristics of enzymatic reactions and may have biological relevance. The complex is in fact capable of catalyzing peroxidative reactions on phenolic compounds related to tyrosine and hydrogen peroxide dismutation. Kinetic and mechanistic studies confirm that the low efficiency with which peroxidative processes occur depends on the limited rate of the reaction between hydrogen peroxide and the iron(III) center, to form the active species, and by the competitive peroxide degradation reaction.

Pancreatic infection in severe pancreatitis: the role of fungus and multiresistant organisms.

HYPOTHESIS: Recent controlled clinical studies suggest a positive effect of early antibiotic treatment on late morbidity and mortality in severe acute pancreatitis. However, widespread use of antibiotics may lead to an increased number of fungal infections and multiresistant bacteria, thereby worsening the outcome of the disease. DESIGN: Single-center prospective study. SETTING: University hospital, gastrointestinal surgical service. PATIENTS: One hundred three patients with necrotizing pancreatitis seen consecutively in our service. INTERVENTIONS: In addition to standard treatment, patients with proven necrotizing pancreatitis received a prophylactic intravenous antibiotic treatment. Pancreatic infection was regarded as an indication for surgery. MAIN OUTCOME MEASURES: Pancreatic infection, microbiological findings, drug resistance, fungal infections. RESULTS: Thirty-three patients (32%) had infected necrosis. Gram-negative organisms were isolated from 19 patients (58%), Gram-positive organisms were isolated from 18 patients (55%), fungal organisms were isolated from 8 patients (24%), and multiresistant organisms were isolated from 3 patients (9%). In 7 patients (21%), the organisms cultured from the pancreatic tissue were resistant to the antibiotics given in for prophylaxis. Infection with multiresistant organisms or organisms resistant to the antibiotic used for prophylaxis, but not with fungal infection or Gram-positive or Gram-negative infection, was correlated with a negative outcome. CONCLUSIONS: Fungal infection under adequate treatment is not associated with a negative outcome. The occurrence of multiresistant organisms seems to be a rare finding (3 of 103 patients). Antibiotic prophylaxis is effective in preventing infection in necrotizing pancreatitis, but optimal choice and duration of administration of the antibiotic agent(s) need to be carefully determined to avoid the sequelae of multiresistant organisms.

Effect of preoperative biliary drainage on surgical outcome after pancreatoduodenectomy.

BACKGROUND: The role of preoperative biliary drainage in patients with biliary obstruction undergoing pancreatoduodenectomy remains controversial. Several authors failed to show any effect of preoperative biliary drainage, whereas others even reported an increased morbidity following pancreatoduodenectomy. METHODS: Retrospective analysis was performed in a consecutive series of 257 patients undergoing pancreatoduodenectomy between November 1993 and November 1999. RESULTS: Ninety-nine patients (38%) underwent preoperative biliary drainage for a median time period of 10 days (range 1 to 41) prior to resection. Cumulative postoperative morbidity was 47% (120 patients), the reoperation rate was 4.3% (11 patients), and mortality was 2.3% (6 patients). There was no difference in total morbidity, infectious complications, reoperation rate, mortality, or long-term survival between patients with or without preoperative biliary drainage. CONCLUSIONS: Preoperative biliary instrumentation and biliary drainage do not affect early or late outcome in patients undergoing pancreatoduodenectomy.

Hyperthermia-induced HSP expression correlates with improved rat renal isograft viability and survival in kidneys harvested from non-heart-beating donors.

Transient sublethal hyperthermia followed by recovery from heat stress, referred to as heat shock preconditioning, exerts a protective effect on ischemia/reperfusion-induced injury in many systems. This effect is considered to be correlated to heat shock proteins (HSPs) and might be a critical factor in kidney graft function and survival. This study was designed to examine the impact of heat shock preconditioning on kidney isograft function and survival in a model utilizing non-heart-beating (NHB) donors. Four groups of male Lewis rats (n = 10/group) subjected either to whole body hyperthermia (groups A and C) or to sham anesthesia (groups B and D) were allowed 24 h recovery. Thereafter, 20 min of warm ischemia (A/B), and in a separate set of experiments 40 min of warm ischemia (C/D), were induced by suprarenal aortic cross clamping before renal procurement. After 24-h preservation with University of Wisconsin solution at 4 degrees C, orthotopic kidney transplantations were performed to syngeneic bilaterally nephrectomized recipients. Tissue specimens were taken to determine HO-1/HSP32, 72, and 90 induction by Western blot analysis. Renal function was measured by means of serum creatinine and creatinine clearance on days 0, 3, and 7 as well as urine volume, protein content, and creatinine levels daily. HO-1/HSP32 and HSP72 were found to be expressed constitutively. Moreover, heat shock strongly induced renal HSP72 and HSP32/HO-1, and to a lesser extent HSP90, expression. For recipients of group A grafts, the graft survival rate was 10/10, whereas it was 7/10 (70 %) in recipients of group B grafts (log rank p < 0.05). Following 40 min of warm ischemia, 6/10 (60 %) recipients survived, whereas all sham treated animals died with anuria within 6 days (log rank p = 0.01). Heat shock preconditioning strongly improved graft viability and reduced functional impairment. Creatinine clearance (CRC) on day 3 post Tx was 0.43 +/- 0.24 ml/min in preconditioned animals (group A) and 0.07 +/- 0.09 ml/min (p < 0.001) in sham preconditioned (group B), whereas it was 0.91 +/- 0.33 ml/min and 0.03 +/- 0.02 ml/min (p < 0.00 001) on day 7 post Tx. Following 40 min NHB time, CRC in survivors of preconditioned graft recipients (group C) was 0.32 +/- 0.2 ml/min (day 3 post Tx) and 0.23 +/- 0.08 ml/min (day 7 post Tx) and was significantly better than CRC of group B (p < 0.01 and p < 0.00001, respectively). CRCs prior to NHB procedures were comparable in all animals ranging between 1.31 and 1.72 ml/min. Serum creatinine as well as proteinuria were significantly increased after transplantation in both groups but recovered within 5 days in recipients of preconditioned grafts, whereas kidneys from donors without HP did not recover function. Histological alterations were also diminished following HP. Hyperthermic preconditioning induces strong and long lasting HO-1/HSP32, HSP72, and HSP90 expression in rat kidneys. HP increases survival following transplantation and improves renal graft function including proteinuria, volume output, and creatinine clearance. HSP induction might be used to develop novel approaches in clinical transplantation.

Liver nodules resembling focal nodular hyperplasia after hepatic venous thrombosis.

We report the case of focal nodular hyperplasia-like nodular hepatic lesions, that developed in the liver of a 35-year-old Caucasian female who required orthotopic liver transplantation for Budd-Chiari syndrome. The rapid development of focal nodular hyperplasia-like lesions in a severe hepatic vascular disorder and in the absence of cirrhosis may represent an additional argument in favor of the vascular origin of focal nodular hyperplasia. The pathogenesis of the nodules is not clear, but pathological arterialization of the liver in hepatic vein thrombosis may be a candidate mechanism.

[Stent or surgical bypass as palliative therapy in obstructive jaundice]. / Stent oder chirurgischer Bypass als palliative Therapie beim Verschlussikterus.

UNLABELLED: During the last decades, the mortality following pancreatic resections has decreased tremendously due to advances in operative technique and perioperative management. In order to examine if similar improvements have been achieved for surgical palliation of obstructive jaundice, we conducted an analysis of our series of surgical bypass procedures. METHODS: Data from all patients undergoing surgical palliation after exploration for pancreatic carcinoma, were prospectively recorded. RESULTS: Between 1.11.93 to 1.11.99 a total of 348 patients were treated with a tumor of the pancreas. 74 of these patients received a bypass procedure: there were 40 double bypass, 20 biliary and 14 gastric bypass procedures. Overall morbidity and mortality was 35% and 1.2% respectively. Median in-hospital stay was 12 days (range 6-37). Median survival time was 5 months (range 1-25). Neither the type of surgical palliation, age nor perioperative risk assessment according to the ASA classification affected perioperative mortality. In contrast, jaundiced patients had significantly more postoperative complications than non-jaundiced patients (58% versus 18%; p = 0.001). CONCLUSIONS: Surgical palliation can nowadays be performed with great safety. A double bypass procedure consisting of a hepatojejunostomy combined with a gastrojejunostomy seems to be the procedure of choice for patients with unresectable pancreatic carcinoma.

Intravenous glucose infusion in labor does not affect maternal and fetal acid-base balance.

OBJECTIVE: This prospective randomized trial compares the effects of a 5% glucose solution or no infusion during labor on glucose levels, pH, pO2, pCO2, and base excess (BE) of normal pregnant women in early labor and at delivery, and on fetal cord blood. METHODS: Forty-three women were randomized to glucose infusion and 38 were controls. RESULTS: Starting glucose levels were independent from the fasting state. When no glucose supplementation was given, the labor itself was associated with a reduction of mean pH (from 7.42 to 7.36, P = 0.00001), mean pCO2 (from 25.7 to 24.4 mm Hg, P = 0.04), and mean BE (from -6.3 to -9.8 mEq/L, P = 0.00001), and an increase of capillary glucose (from a mean of 83 to 105 mg/dL, P = 0.00001). Infusions of glucose did not significantly alter maternal acid-base balance at delivery. pH, PO2, pCO2, and BE were similar in arterial and venous cord blood of both groups. No variables correlated with cord blood glucose levels or with glucose vein-artery difference. CONCLUSIONS: We conclude that a 5% glucose infusion does not significantly reduce maternal acidemia associated with vaginal delivery and therefore its use cannot be recommended, since maternal glucose is largely available during labor. Intrapartum glucose infusions do not alter the acid-base balance, when the fetus is well oxygenated.