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不同病因肝硬化患者血清瘦素水平的差异

【摘要】  目的 探讨不同病因肝硬化患者血清瘦素水平的差异。方法 采用放射免疫法测定研究对象血清瘦素水平,并比较不同病因肝硬化患者血清瘦素水平的差异。结果 各肝硬化组血清瘦素的水平明显高于正常对照组(P<0.01)。乙肝肝硬化组与丙肝肝硬化组相比较,瘦素水平无明显差异(P>0.05);酒精性肝硬化组与非酒精性脂肪性肝硬化组相比较,瘦素水平无明显差异(P>0.05);酒精性肝硬化组与非酒精性脂肪性肝硬化组瘦素水均高于乙肝肝硬化组与丙肝肝硬化组(P<0.05)。结论 肝硬化患者血清瘦素水平明显升高,酒精性肝硬化与非酒精性脂肪性肝硬化患者血清瘦素水平升高更加明显,其原因除肝星状细胞(HSC)激活外,与存在瘦素抵抗有关。

【关键词】  肝硬化;血清瘦素;酒精性肝硬化

The difference of serum leptin in different types of hepatic cirrhosis patients

    LI Kun1  ZHENG Tielong2

    1 Department of Degestive Disease,Affiliatied Hospital of Binzhou Medical University,

    Binzhou,256603;2 Institute of Infectious Disease,Ditan Hospital,Beijing 100011

    【Abstract】  Objective  To study the difference of serum leptin in different types of hepatic cirrhosis patients.Methods  Detect the serum leptin of the patients through radioimmunity,and compare the difference of serum leptin in different types of hepatic cirrhosis patients.Results   The serum leptin in all types of hepatic cirrhosis was obviously higher than that in normal control(P<0.01). There was no significant difference between the serum leptin in hepatitis B cirrhosis and hepatitis C cirrhosis patients (P>0.05). There was no significant difference between the serum leptin in alcoholic hepatic cirrhosis and nonalcoholic fatty cirrhosis patients (P>0.05).The serum leptin in alcoholic cirrhosis and nonalcoholic fatty hepatic cirrhosis patients was obviously higher than that in hepatitis B cirrhosis and hepatitis C cirrhosis patients (P<0.05).Conclusion  The serum leptin in cirrhosis patients increased obviously.The serum leptin in alcoholic hepatic cirrhosis and nonalcoholic fatty hepatic cirrhosis patients increased more obviously because of the activation of HSC and the leptin resistance.

    【Key words】  hepatic cirrhosis,serum leptin,alcoholic cirrhosis

    瘦素(leptin) 是由167 个氨基酸组成的一种调节能量平衡的多肽激素,是肥胖基因(ob gene) 编码的产物[1]。近年来的研究发现,瘦素与多种慢性肝病的发生和发展密切相关,尤其与肝硬化的关系日益受到重视。本研究测定了不同病因肝硬化患者血清瘦素水平,并探讨不同病因肝硬化患者血清瘦素水平差异的原因。

    1  对象与方法

    1.1  对象选取  住院肝硬化患者58例,均为男性,平均年龄42岁。其中乙肝肝硬化25例,丙肝肝硬化13例,酒精性肝硬化12例,非酒精性脂肪性肝硬化8例,病例选择依据2000年修订病毒性肝炎防治方案中肝硬化的诊断标准[2]及2006年酒精性肝病诊疗指南与非酒精性脂肪性肝病诊疗指南[3,4]。对照组30例为男性健康体检者,平均年龄40岁。所有肝硬化患者均无肾脏功能损害,无感染,无肿瘤或代谢性疾病,至少1年内未服用糖皮质激素或免疫抑制剂;对照组排除重要器官疾病、肿瘤、感染和代谢性疾病,至少1年内未服用糖皮质激素或免疫抑制剂。

    1.2  标本采集  隔夜空腹8 h抽取肘静脉血6 ml,2 h内分离血清后,置于-80℃冰箱内保存待测。

    1.3  测定方法  受检者均测定肝功能、瘦素。瘦素测定采用放射免疫法,试剂盒购于美国Linco公司,肝功能采用全自动生化仪测定。

    1.4  统计学分析  采用SPSS12.0软件进行数据统计分析,所有结果均用x±s表示。各试验组与对照组比较采用Dunnett检验,组间比较采用KruskalWallis检验。

    2  结果

    2.1  各肝硬化组与对照组参数比较  各肝硬化组血清瘦素的水平明显高于正常对照组(P<0.01),见表1。

    2.2  各肝硬化组参数相互比较  乙肝肝硬化组与丙肝肝硬化组相比较,瘦素水平无明显差异(P>0.05);酒精性肝硬化组与非酒精性脂肪性肝硬化组相比较,瘦素水平无明显差异(P>0.05);酒精性肝硬化组与非酒精性脂肪性肝硬化组瘦素水均高于乙肝肝硬化组与丙肝肝硬化组(P<0.05),见表1。表1  各肝硬化组与对照组血清瘦素水平比较各肝硬化组血清瘦素水平明显高于正常对照组,P<0.01;酒精性肝硬化组与非酒精性脂肪性肝硬化组瘦素水平均高于乙肝肝硬化组与丙肝肝硬化组,P<0.05。

    2.3  按肝功能分级患者血清瘦素水平的差异  按ChildPugh标准进行肝功能分级,由A级至C级,血清瘦素水平逐步升高,差异有显著性 (P<0.05),见表2。表2  按肝功能分级血清瘦素的水平肝功能

    3  讨论

    瘦素作为肥胖基因的产物,主要作用是调节体内能量代谢和脂肪沉积。自Potter 等[5]发现活化的肝星状细胞(HSC)能合成瘦素以来,瘦素与肝脏疾病的关系正日益受到重视。HSC 是肝纤维化时细胞外基质的主要来源,其激活是肝纤维化的中心环节,在肝硬化发展进程中起关键作用。近期研究发现活化的HSC 胞质中有瘦素mRNA 表达和瘦素蛋白合成,瘦素通过与其受体(obR) 结合发挥作用,促进胶原等ECM 的合成、分泌,从而促进肝纤维化[6]。多数研究证实,肝硬化患者的血清瘦素水平显著高于正常对照组,随着肝功能的恶化,血清瘦素水平呈上升趋势[7,8]。而Greco等[9]的研究则显示肝硬化患者血清瘦素水平较正常对照组降低,且随肝功能的恶化而呈下降趋势。

    本研究测定了不同病因肝硬化患者血清瘦素水平证实,肝硬化患者血清瘦素水平明显高于正常对照组,且随着肝功能的恶化,血清瘦素水平呈上升趋势。酒精性肝硬化与非酒精性脂肪性肝硬化患者血清瘦素水均高于乙肝肝硬化与丙肝肝硬化患者。该结果提示,酒精性肝硬化与非酒精性脂肪性肝硬化患者瘦素水平明显升高,除HSC激活之外,尚存在其他导致瘦素水平升高的因素。

    酒精性肝硬化与非酒精性脂肪性肝硬化患者血清瘦素水平均明显升高,而且瘦素的有效生物学效应明显下降,提示存在瘦素抵抗。瘦素抵抗使正常的胰岛脂肪细胞轴反馈机制受到破坏,瘦素抑制胰岛素分泌的能力降低,加重了机体的胰岛素抵抗及高胰岛素血症,导致肝脏摄取脂肪增加、肝细胞色素P4502EI (CYP2EI) 表达增加、肝细胞损伤或诱导中性粒细胞和其他炎症细胞的聚集和浸润[10]。高水平的内源性瘦素不仅无助于肥胖患者的体重控制,还可引起胰岛素抵抗,刺激巨噬细胞分泌肿瘤坏死因子α及IL6 、IL12,并促进肝星状细胞分化及内脏脂肪积聚,从而促进单纯性脂肪肝向脂肪性肝炎及肝纤维化、肝硬化转变[11]。瘦素抵抗可能参与了肝脏脂肪变性的形成[12,13]。Petersen等[14]发现,具有严重胰岛素抵抗和脂质代谢异常的脂肪肝病人应用瘦素治疗后,肝脏胰岛素敏感性增加,肝脏内的TG含量减少约85 % ,可逆转胰岛素抵抗和肝脏脂肪变性。对于酒精性肝硬化与非酒精性脂肪性肝硬化患者,这可能是一种新的、可行的治疗方法。

【参考文献】
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[2] 中华医学会传染病与寄生虫学分会、肝病学分会.病毒性肝炎防治方案[J].中华肝脏病学杂志,2000,8(6):324.

[3] 中华医学会肝病学分会脂肪肝和酒精性肝病学组. 酒精性肝病诊疗指南[J].中华肝脏病杂志,2006,14(3):164.

[4] 中华医学会肝病学分会脂肪肝和酒精性肝病学组. 非酒精性脂肪性肝病诊疗指南[J].中华肝脏病杂志,2006,14(3):161.

[5] Potter JJ, Womack L, Mezey E,et al.Transdifferentiation of rat hepatic stellate cells results in leptin expression[J].Biochem Biophys Res Commun, 1998,244(1): 178.

[6] Ikejima K,Honda H, Yoshikawa M,et al. Leptin auguments inflammatory and profibrogenic responses in the murine liver induced by hepatoxic chemicals[J].Hepatology ,2001 ,34 (2) :288.

[7] Lin SY, Wang YY, Sheu WH. Increased serum leptin concentrations correlate with soluble tumour necrosis factor receptor levels in patients with cirrhosis[J].Clin Endocrinol (Oxf), 2002, 57(6): 805.

[8] Testa R, Franceschini R, Giannini E,et al.Serum leptin levels in patients with viral chronic hepatitis or liver cirrhosis[J].J Hepatol, 2000, 33(1): 33.

[9] Greco AV, Mingrone G, Favuzzi A,et al.Serum leptin levels in posthepatitis liver cirrhosis[J].J Hepatol, 2000, 33(1): 38.

[10] 马向华,王维敏. 非酒精性脂肪肝患者瘦素抵抗和胰岛素抵抗研究[J].中华肝脏病杂志,2004 ,12(11)∶651.

[11] Gunel N , Coskun U,Toruner FB ,et al. Serum leptin levels are associated with tamoxifen induced hepatic steatosis[J].Curr Med Res Opin ,2003 ,19(1)∶47.

[12] Campillo B , Sherman E , Richardet JP,et al.Serum leptin levels in alcoholic liver cirrhosis : relationship with gender nutritional status , liver function and energy metabolism[J].Eur J Clin Nur , 2001 ,55 (11) :980.

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[14] Petersen KF , Oral EA , Dufour S,et al.Leptin reverses insulin resistance and hepatic steatosis in patients with severse lipodystrophy[J].J Clin Invest , 2002 , 109 ( 10 ) :1345.


作者单位:1 滨州医学院附属医院消化内科 滨州市 256603;2 北京地坛医院传染病研究所

日期:2009年8月25日 - 来自[2009年第32卷第1期]栏目

Altered postprandial glucose, insulin, leptin, and ghrelin in liver cirrhosis: correlations with energy intake and resting energy expenditure

Evangelos Kalaitzakis, Ingvar Bosaeus, Lena Öhman and Einar Björnsson

1 From the Department of Internal Medicine, Faculty of Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden (EK, IB, and EB), and the Department of Clinical Immunology, Faculty of Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden (LÖ)

2 Supported by the Faculty of Medicine, University of Gothenburg.

3 Reprints not available. Address correspondence to E Kalaitzakis, Department of Internal Medicine, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden. E-mail: evangelos.kalaitzakis{at}vgregion.se.


ABSTRACT  
Background:Liver cirrhosis is associated with reduced energy intake and increased resting energy expenditure.

Objective:We aimed to investigate the possible role of glucose, insulin, leptin, and ghrelin in the pathogenesis of these alterations.

Design:Nutritional status, energy intake, resting energy expenditure, and fasting glucose, insulin, and leptin were assessed in 31 patients with cirrhosis. Postprandial glucose, insulin, C-peptide, leptin, and ghrelin responses were studied in a subgroup of patients after a standard meal. Ten healthy subjects served as controls.

Results:Patients with cirrhosis had a lower energy intake (P < 0.05), higher resting energy expenditure (P < 0.05), higher fasting leptin (P < 0.05), and higher insulin resistance (P < 0.001) than did the healthy control subjects. In the patients with cirrhosis, fasting leptin was negatively correlated with resting energy expenditure (r = –0.38, P < 0.05) but not with energy intake. In control subjects, leptin was negatively correlated with energy intake (r = –0.72, P < 0.05) but not with resting energy expenditure. The patients with cirrhosis had higher postprandial glucose (P < 0.001) and lower ghrelin (P < 0.05) concentrations at 4 h postprandially than did the control subjects. The increase in ghrelin from its minimal postmeal value to 4 h postmeal was negatively correlated (r = –0.66, P = 0.014) with weight loss in the patients with cirrhosis. Energy intake was negatively correlated (r = –0.42, P < 0.01) with the postprandial increase in glucose.

Conclusions:In cirrhosis, altered postprandial glucose and ghrelin are associated with reduced energy intake and weight loss, respectively, and the effects of leptin on energy intake and expenditure seem to be altered. Insulin resistance might be involved in these altered postprandial responses.

Key Words: Glucose • insulin • leptin • ghrelin • insulin resistance • energy intake • resting energy expenditure • liver cirrhosis • malnutrition


INTRODUCTION  
Malnutrition is common in patients with liver cirrhosis, with a reported prevalence as high as 80% depending on the severity of liver disease (1-3). The mechanisms of malnutrition in cirrhosis are not completely understood. Both poor dietary intake (3-5) and increased basal energy expenditure have been reported to contribute to a negative energy balance in patients with cirrhosis (1, 6-11).

Insulin resistance is common in patients with cirrhosis (3, 7, 12) and is possibly associated with impairment of nutritional status (12). An elevated postprandial insulin concentration has been proposed as a factor that induces satiety and a subsequent reduction in energy intake in liver cirrhosis (12). However, the relation of postprandial hyperglycemia to energy intake, which has been shown to occur in cirrhosis (12), is unexplored in this group of patients.

Leptin and ghrelin are known to influence energy expenditure and energy intake in humans (13). Leptin circulates in free and bound form, and it has been shown to suppress energy intake and stimulate energy expenditure, whereas ghrelin has been shown to rise before a meal thus enhancing appetite and food intake (13). The basal concentrations of leptin and ghrelin have been reported to be deranged in liver cirrhosis (5, 9, 14-19), but only few studies are available on the relations of leptin and ghrelin to energy intake and resting energy expenditure (REE) in these patients (5, 18, 19). In a previous report, no correlation was found between total leptin concentration and REE in patients with cirrhosis with adequate food intake (18). Also, bound (but not free) leptin was shown to be increased and positively correlated with REE in patients with postviral cirrhosis on a weight-maintaining diet (9). To our knowledge, the relation of leptin to spontaneous energy intake and REE in patients with cirrhosis of various etiologies has not been previously investigated. Also, data are lacking on postprandial changes in leptin and ghrelin in patients with cirrhosis.

Insulin has been reported to be essential for meal-induced ghrelin suppression (20-22) and to acutely increase leptin in healthy persons (23). An inverse relation between leptin and ghrelin has been observed, and it has been proposed that leptin could be of importance for suppression of basal ghrelin in normoinsulinemic subjects (24). Thus, to study the potential importance of these hormones for energy intake and REE, they need to be investigated together, a study not previously undertaken in liver cirrhosis.

The main aim of the current study was to investigate the relation of basal and postprandial concentrations of plasma glucose, insulin, leptin, and ghrelin to energy intake and REE. A secondary aim was to study the interrelations of postprandial plasma glucose, insulin, leptin, and ghrelin in patients with cirrhosis.


SUBJECTS AND METHODS  
Thirty-one consecutive patients with liver cirrhosis attending the outpatient clinic of the Department of Internal Medicine at Sahlgrenska University Hospital, Gothenburg, Sweden, were enrolled in the study. The diagnosis of liver cirrhosis was established histologically; on the basis of its clinical, laboratory, endoscopic, or imaging features; or both. The severity of liver disease was assessed according to the Child-Pugh and the Model for End Stage Liver Disease scores (25). Patients with malignancy, infections, known gastrointestinal or renal disease, significant respiratory or cardiac dysfunction, insulin-dependent diabetes mellitus, hepatorenal syndrome, untreated thyroid dysfunction, and hepatic encephalopathy grade II–IV were excluded. Patients with alcoholic cirrhosis had been abstinent for 6 mo at inclusion. All had normal serum creatinine and had undergone gastroscopy in the previous 6 mo. Twenty-six of the 31 patients had endoscopic evidence of esophageal varices, and 20 of the 31 had evidence of portal hypertensive gastropathy. None of the patients had macroscopic evidence of gastric mucosal atrophy. Two patients were found to have diabetes mellitus on blood sampling for purposes of this study. Six patients had mild ascites detectable by ultrasonography at inclusion and were treated with spironolactone. None had peripheral edema. Ten age-, sex-, and body mass index (BMI)–matched healthy weight-stable volunteers, mainly health-care professionals, acted as controls. Most of them had participated in several studies as healthy volunteers before, none was taking any medications, none was obese, all denied alcohol overconsumption, and all had normal liver function tests. The study was approved by the ethics committee of the University of Gothenburg and informed consent was obtained from all subjects.

Assessment of nutritional status
Weight was measured without shoes and in light clothing. Of 6 patients with mild ascites, every effort was made to calculate dry weight, which is defined as body weight after taking into consideration water overload. The dry weight was considered equal to the current weight if no ascites was present. In patients with ascites, a review of the patient files was performed to find data on weight after last paracentesis or before recent ascites development. BMI was calculated and weight change that could not be explained by ascites or edema during the previous 6 mo was noted. Dry weight loss was expressed as a percentage of actual body weight. Skinfold thickness at the tricep, bicep, subscapular, and suprailiac sites as well as midarm muscle circumference were measured 3 times by the same research dietitian, and the mean value was used. The sum of the tricep, bicep, subscapular, and suprailiac skinfolds was used to assess percentage body fat according to previously published age- and sex-specific tables (26). This method has been shown to have comparable results with dual energy X-ray absorptiometry in patients with cirrhosis without overt fluid retention (27). Fat-free mass (FFM) was calculated as body weight minus fat mass. Patients were considered malnourished when the triceps skinfold thickness, midarm muscle circumference, or both were below the 5th percentile, according to standard tables for the Swedish population based on age and sex (28), or if BMI (in kg/m2) was < 18.5.

Dietary intake
To assess the subjects' dietary intake, a 4-d food diary was used as previously described (29). Total daily energy intake is reported in absolute amounts, as a ratio of body weight in kg (energy intake:body weight), and as a ratio of REE (energy intake:REE).

Indirect calorimetry
REE was determined for all subjects in the morning after an overnight fast (10 h) by indirect calorimetry (Deltatrac; Datex, Helsinki, Finland) from 0730 to 0830. To compare REE between the different groups, REE was adjusted for FFM by the use of a linear regression model. Adjusted REE was calculated as the group median REE plus measured REE minus predicted REE, where group median REE is the median absolute REE, measured REE is the metabolic rate measured in each subject, and predicted REE is the calculated rate obtained by using the individual FFM in the linear regression equation generated from the cirrhotic or control group as appropriate (30). Hypermetabolism was defined as a ratio of measured REE to predicted REE > 1.1 (29).

Test meal
On another day, about one week apart from indirect calorimetry, from 0730 to 0800 after an overnight fast, a subgroup of 18 patients with cirrhosis (group A) and all healthy control subjects had a 480 kcal test meal of oatmeal porridge and one cheese sandwich with set amounts of macronutrients (55% of energy as carbohydrate, 31% of energy as fat, and 14% of energy as protein). The test meal is a common kind of breakfast in Scandinavia. The subjects were instructed to eat the meal within 10 min. Blood samples for serum insulin, plasma glucose, and serum C-peptide measurements were drawn from an indwelling cannula at baseline and at 30 min, 60 min, 90 min, 2 h, and 4 h after the meal. In a subgroup of group A—13 patients with cirrhosis (group B)—and all healthy control subjects blood samples were also drawn for plasma leptin and ghrelin analysis at the same intervals.

Blood sample analysis
Blood samples for glucose, insulin, and leptin were drawn after an overnight fast on the day of the test meal from subjects who participated in this part of the study and on the day of indirect calorimetry from all others. Insulin resistance was expressed as homeostasis model assessment index (HOMA-IR) (31). Plasma was immediately separated by centrifugation for 5 min at 1000 x g (4 °C) and then stored at –80 °C until subsequent leptin, ghrelin, or C-peptide analysis. Plasma total ghrelin concentrations were measured by commercial RIA (Linco Research Inc, St Louis, MO) by using 125I-labeled ghrelin as a tracer and ghrelin antiserum specific for total ghrelin. The detection limit for the assay was 93 pg/mL. Ghrelin was expressed in absolute values. Plasma leptin concentrations were measured by using a commercial enzyme-linked immunosorbent assay (Quantikine human leptin, R&D Systems, Oxford, United Kingdom). The detection limit for the assay was 15.6 pg/mL. Leptin was expressed in absolute values and as a ratio of leptin to weight (leptin:body weight), of leptin to BMI (leptin:BMI), and of leptin to fat in kg (leptin:fat). Patients in subgroup B underwent serological testing for the detection of Helicobacter pylori performed according to standard in-house methods.

Statistics
Data are expressed as medians and interquartile ranges (IQRs). The Mann-Whitney U test was performed for calculations of differences between groups. For correlation analysis, the Spearman coefficient was calculated. Partial correlation analysis was performed to control for covariates. The chi-square test was used for comparisons between qualitative variables (sex, presence of diabetes, or hypermetabolism). To evaluate plasma glucose, insulin, leptin, and ghrelin changes postprandially, the Friedman's test was used. When the P value was < 0.05, a post hoc analysis with the Wilcoxon's signed rank test was performed. Multivariate repeated-measures analysis of variance was used to test the interaction between time and group. When the P value was < 0.05, the Mann-Whitney U test was used to compare the 2 groups at each time point. Stepwise linear regression analysis was used to determine the correlation of independent variables with the energy intake:body weightor the area under the glucose curve (dependent variables), which were transformed into a normal score by using the Blom's method. All tests were two-tailed and conducted at a 5% significance level. Statistical analysis was done by using SPSS version 11.0.2 (SPSS Inc, Chicago, IL).


RESULTS  
The basic characteristics of the patients and healthy control subjects are shown in Table 1. The patients with cirrhosis had higher insulin resistance, leptin, and REE (adjusted for FFM) as well as lower energy intake than did the healthy control subjects (Table 2). No significant differences in any of the variables in Table 2 were observed between the patients with alcoholic and those with nonalcoholic cirrhosis, the patients with Child-Pugh class A and those with Child-Pugh class B or C, the patients with malnutrition and those without malnutrition, and the patients with hepatic encapholopathy and those without hepatic encephalopathy (data not shown).


View this table:
TABLE 1. Basic characteristics in all subjects1

 

View this table:
TABLE 2. Metabolic and dietary data in patients with cirrhosis and healthy control subjects1

 
Fasting leptin was positively correlated with BMI in patients with cirrhosis (r = 0.48, P = 0.007). Also, leptin was positively correlated with body fat (in kg) in the healthy control subjects (r = 0.78, P = 0.008) but not in patients with cirrhosis (r = 0.18, P = 0.4). After control for BMI (partial correlation analysis), fasting leptin was positively correlated with HOMA-IR (r = 0.4, P = 0.034), negatively correlated with REE (r = –0.38, P = 0.042), and not significantly correlated with energy intake (r = –0.04, P = 0.8) in patients with cirrhosis. After control for BMI (partial correlation analysis), fasting leptin was negatively correlated with energy intake (r = –0.72, P = 0.029) but not to HOMA-IR (r = –0.48, P = 0.2) or REE (r = –0.49, P = 0.2) in control subjects.

Postprandial glucose
At 30 min postprandially, plasma glucose had risen in both the cirrhosis and the control groups but subsequently remained elevated only in the former (Figure 1). The interaction between time and group for glucose was found to be significant (P = 0.037). The area under the glucose curve (AUC) and the increase of glucose from baseline to 60 min postprandially were higher in the patients with cirrhosis than in the control subjects [respective median (IQR) AUCs: 13.7 mmoll–1h–1 (11.9–15) compared with 10.9 mmoll–1h–1 (8.8–11.2); P < 0.001; and respective median (IQR) increases: 54.8% (22.1–79.6%) compared with 20% (–21.3% to 31.9%); P = 0.002, respectively]. The increase of glucose from baseline to 60 min postprandially was negatively correlated with the ratio of energy intake to body weight in the patients with liver cirrhosis (r = –0.53, P = 0.023) but not in the healthy control subjects (r = 0.37, P = 0.3). HOMA-IR was positively correlated with the AUC of glucose in the patients with cirrhosis (r = 0.75, P < 0.001) but not in the control subjects (r = 0.16, P = 0.7).


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FIGURE 1.. Median (half interquartile range) postprandial plasma glucose, serum insulin, and serum C-peptide concentrations and molar ratio of serum insulin to C-peptide in patients with cirrhosis (solid line; n = 18) and in healthy control subjects (dashed line; n = 10). Glucose changed significantly in patients with liver cirrhosis (Friedman's test, P = 0.001) and in healthy control subjects (Friedman's test, P = 0.001). The interaction between time and group was significant (P = 0.037). Insulin changed significantly in both the patients with liver cirrhosis and the healthy control subjects (Friedman's test, P = 0.001 for both). The interaction between time and group was not significant. C-peptide changed significantly in both the patients with liver cirrhosis and the healthy control subjects (Friedman's test, P = 0.001 for both). The interaction between time and group was significant (P = 0.036). The molar ratio of insulin to C-peptide changed significantly in both patients with liver cirrhosis and in healthy control subjects (Friedman's test, P = 0.001 for both). The interaction between time and group was not significant. Significantly different from baseline values, P = 0.05 (Wilcoxon's signed rank test). #Significantly different from the healthy control subjects, #P = 0.05 (Mann-Whitney U test).

 
Postprandial insulin
At 30 min, serum insulin had risen in both the patients with cirrhosis and the control subjects and remained elevated until 2 h postmeal in both groups (Figure 1). The interaction between time and group for insulin was not significant.

Postprandial C-peptide and serum insulin-to-C-peptide molar ratio
The interaction between time and group for C-peptide was significant (P = 0.035). The postprandial C-peptide response was higher in the patients with liver cirrhosis than in the healthy control subjects (Figure 1; AUC of C-peptide: 4.9 nmoll–1h–1 (IQR: 4.2–6.7 nmoll–1h–1) compared with 2.6 nmoll–1h–1 (2.4–3.5 nmoll–1h–1; P < 0.001). The postprandial insulin-to-C-peptide molar ratio response, a measure of portosystemic shunting, in patients with cirrhosis and healthy control subjects is shown in Figure 1. The interaction between time and group for the insulin-to-C-peptide molar ratio was not significant.

Postprandial leptin
The interaction between time and group for leptin was not significant (Figure 2). Similar results were obtained when leptin:BMI, leptin:body weight, or leptin:fat in kg were used instead of uncorrected leptin values.


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FIGURE 2.. Median (half interquartile range) postprandial plasma leptin and ghrelin concentrations in patients with cirrhosis (solid line; n = 13) and in healthy control subjects (dashed line; n = 10). Leptin changed signficantly in patients with liver cirrhosis (Friedman's test, P = 0.001) but not in healthy control subjects (Friedman's test, P = 0.5). The interaction between time and group was not significant. Ghrelin changed significantly in the healthy control subjects (Friedman's test, P = 0.001) but not in the patients with liver cirrhosis (Friedman's test, P = 0.13). The interaction between time and group was significant (P = 0.015). Significantly different from concentrations at 4 h, P = 0.01 (Wilcoxon's signed-rank test). #Significantly different from healthy control subjects, P = 0.021 (Mann-Whitney U test).

 
Postprandial ghrelin
Postprandial ghrelin changed significantly compared with baseline only in the healthy control subjects (Figure 2). The interaction between time and group for ghrelin was significant (P = 0.015). At 4 h, ghrelin was higher in the healthy control subjects than in the patients with liver cirrhosis [1176 pg/mL (IQR: 679.3–1692 pg/mL) compared with 519 pg/mL (379.5–607 pg/mL); P = 0.021]. The increase of ghrelin from its minimal postmeal value to 4 h postmeal was higher in the healthy control subjects than in the patients with cirrhosis [39% (33.1–48.2%) compared with 14.2% (12.8–33.4%); P = 0.005], and it was negatively correlated with weight loss in the previous 6 mo in the patients with cirrhosis (r = –0.66, P = 0.014). The AUC of ghrelin did not differ significantly between the patients with cirrhosis and the healthy control subjects (data not shown). Postprandial ghrelin concentrations were negatively correlated with glucose and insulin in both the patients with liver cirrhosis and the healthy control subjects (Table 3). The postprandial ghrelin decrease was positively correlated with leptin decrease in the healthy control subjects and negatively in the patients with liver cirrhosis (Table 3). Ghrelin concentrations were not significantly different at any time point between the patients with and those without portal hypertensive gastropathy and between the patients with (n = 3) and those without (n = 10) serological positivity for Helicobacter pylori (data not shown).


View this table:
TABLE 3. Spearman correlations of postprandial ghrelin with postprandial glucose, insulin, and leptin variables in patients with liver cirrhosis and healthy control subjects1

 
Regression analysis
Stepwise linear regression analysis was performed for the cirrhosis group with the ratio of energy intake to body weight as the dependent variable. Child-Pugh score, REE, the increase in glucose 60 min postprandially, and the increase in ghrelin from its minimal postmeal value to 4 h postmeal were used as independent variables. Only the increase in glucose 60 min postprandially was found to be independently correlated with energy intake (ß = –0.42, P = 0.019).

In an attempt to identify factors involved in the increased postprandial glucose response, stepwise regression analysis was also performed for the cirrhosis group with AUC of glucose as the dependent variable. Percentage fat mass, HOMA-IR, baseline glucose concentrations, the Child-Pugh score, and the fasting serum insulin-to-C-peptide molar ratio (as a measure of hepatic shunt volume) were used as independent variables. Only insulin resistance expressed as HOMA-IR was found to be independetly correlated with the postprandial glucose response (ß = 0.82, P = 0.001) in the patients with cirrhosis.


DISCUSSION  
In the current study, we observed altered postprandial responses of glucose and ghrelin associated with reduced energy intake and weight loss in patients with liver cirrhosis. The patients with cirrhosis exhibited insulin resistance with higher baseline and postprandial glucose concentrations compared with the healthy control subjects, which agrees with the results of previous studies (3, 7, 12). Although the patients with cirrhosis exhibited both higher fasting insulin and C-peptide concentrations than did the control subjects, indicating increased insulin production in the cirrhotic subjects, the postprandial glucose response was found to be independently related only to insulin resistance. Furthermore, the postprandial increase in glucose was found to contribute independently to the reduced energy intake in the patients with cirrhosis. Decreased hunger and slower gastric emptying were observed in healthy volunteers during induced hyperglycemia (32). Postprandial hyperglycemia has been reported to be associated with increased postprandial upper gastrointestinal symptoms (33, 34) compared with euglycemia in healthy volunteers. We recently reported an increased prevalence of gastrointestinal symptoms (including early satiety) in patients with cirrhosis (35, 36). It is therefore possible that postprandial hyperglycemia results in reduced energy intake by contributing to early satiety and other gastrointestinal symptoms in patients with cirrhosis.

Baseline leptin in patients with cirrhosis was found to be elevated, as previously reported (14-18), and leptin effects on energy intake and REE were disturbed in these patients. Leptin has been shown to increase REE (13), but in a recent study performed in non-cirrhotic individuals, total and free leptin were reported to be negatively and bound leptin positively associated with REE (37). We observed a negative association between total leptin and REE in patients with cirrhosis. It might therefore be hypothesized that the resistance to the effects of leptin in cirrhotics observed in the current study is mediated by a proportional increase in free leptin. However, we did not measure free and bound leptin fractions in our series, which is mandatory to show this. Alternatively, the disturbed associations of leptin with energy intake and REE in cirrhosis might simply indicate disturbed metabolic regulation in these patients, documenting the central role of liver metabolism in whole-body fuel homeostasis. The results of the current study, however, do not support a role of postprandial leptin concentrations in the low energy intake seen in patients with cirrhosis.

Ghrelin concentrations after a meal have not been investigated previously in patients with liver cirrhosis. The patients with cirrhosis had a clearly altered postprandial pattern of ghrelin compared with the control subjects, with an attenuated ghrelin increase at 4 h postmeal. Ghrelin enhances appetite and food intake, and its concentration rises preprandially, thus playing a role in meal initiation (13). Therefore, the low ghrelin observed in the patients with cirrhosis at 4 h postmeal (ie, before expected lunch in our experiment setting) could be involved in the reduced energy intake in these patients. In a recent study, fasting ghrelin was found to be elevated in patients with liver disease compared with healthy control subjects (19). Marchesini et al (5) reported that fasting ghrelin was comparable in patients with cirrhosis and control subjects but increased concentrations were identified in a group of patients with low energy intake and malnutrition. In our study, we were also unable to confirm generally increased fasting ghrelin in patients with cirrhosis. These discrepancies could, at least in part, be explained by different patient selection, control subject selection, or both. Patients in the former study (19) were transplantation candidates, some had malignancies and were not BMI-matched with control subjects, whereas in the current study, no patients with malignancies were included and BMI-matched control subjects were chosen.

The mechanisms of altered postprandial ghrelin response might involve glucose, insulin, leptin, or all three. Postprandial ghrelin was negatively related to glucose and insulin in both healthy control subjects and patients with cirrhosis, as previously reported (20-22). According to these studies, insulinemia is essential for postprandial ghrelin suppression with glucose having an additional effect (20-22). In our series, the postprandial ghrelin decrease was negatively related to leptin reduction in the patients with cirrhosis. This agrees with earlier data suggesting an inverse relation between leptin and ghrelin and that leptin could be important for suppression of ghrelin (24). Therefore, insulin resistance resulting in high postprandial glucose and insulin might be involved in the low ghrelin observed 4 h postmeal. Thus, it is conceivable that treatment of insulin resistance might reduce the hypoghrelinemia before a meal in patients with cirrhosis, possibly stimulating appetite. Although this is probably not the single most important reason for reduced energy intake in liver cirrhosis, it certainly warrants further studies.

Certain methodologic aspects should be taken into consideration when interpreting the results of the current study. Food intake was assessed by means of food diaries. This is an established method of food intake assessment (29, 37-39), which has been previously utilized in patients with liver cirrhosis (4, 5, 12). However, it is known that both normal-weight and obese subjects may underestimate their dietary intake (39), and it is conceivable that patients with hepatic encephalopathy might also be prone to underreporting when filling in detailed food diaries. In the current study, no patients with encephalopathy grade II or higher were included and food intake was not statistically different between the patients with and those without hepatic encephalopathy grade I. Furthermore, our findings confirm previous studies showing reduced energy intake in patients with cirrhosis (3-5) and reports of a negative correlation between leptin and food intake in healthy subjects (39). Second, in the current study, fasting data were obtained from all subjects but postprandial data were obtained from a smaller subgroup of the main patient population. Although the patients with cirrhosis were carefully matched with the group of healthy control subjects, a type 2 error in the assessment of the postprandial responses cannot be ruled out. Lastly, the current study was a cross-sectional one. Thus, statistical correlations between hormonal disturbances and energy intake or REE in cirrhosis do not necessarily implicate a cause-effect relation.

In conclusion, altered postprandial glucose and ghrelin concentrations correlated with reduced energy intake and weight loss in liver cirrhosis. The effects of leptin on energy expenditure and energy intake seem to be altered in patients with cirrhosis. Insulin resistance might be involved in the altered postprandial glucose and ghrelin responses.


ACKNOWLEDGMENTS  
We thank RN Pernilla Jerlstad and dietitian Stine Storsrud for expert technical assistance.

EK contributed to the design of the study, collection and analysis of data, and writing of the manuscript. IB provided advice and consultation on the design of the study and on the writing of the manuscript as well as final review and approval. LÖ contributed to the analysis of the data and reviewed and approved the final manuscript. EB contributed to the design of the study and writing of the manuscript. None of the authors have a personal or financial conflict of interest.


REFERENCES  

Received for publication March 8, 2006. Accepted for publication November 3, 2006.


日期:2008年12月28日 - 来自[2007年85卷第3期]栏目

Absorption and transport of dietary long-chain fatty acids in cirrhosis: a stable-isotope-tracing study

Eduard Cabré, José M Hernández-Pérez, Lourdes Fluvià, Cruz Pastor, August Corominas and Miquel A Gassull

1 From the Departments of Gastroenterology (EC and MAG) and Biochemistry (JMH-P, LF, CP, and AC), Hospital Universitari Germans Trias i Pujol, Badalona, Catalonia, Spain

2 Supported by grants from the Fondo de Investigación Sanitaria (FIS 96/1368) and Instituto de Salud Carlos III (C03/02) of the Spanish Government. JMH-P received a grant from the Fondo de Investigación Sanitaria of the Spanish Government (BAE 98/5066) and the Fundació per a la Recerca Biomèdica Germans Trias i Pujol.

3 Address reprint requests to MA Gassull, Department of Gastroenterology, Hospital Universitari Germans Trias i Pujol, Carretera de Canyet s/n, 08916 Badalona, Catalonia, Spain. E-mail: mgassull{at}ns.hugtip.scs.es.


ABSTRACT  
Background: In rats, 30–70% of dietary fatty acids (FAs) are absorbed through the portal vein. Whether this occurs in humans is unknown, but it may occur in persons with cirrhosis, who show a blunted chylomicronemic response to dietary fat without significant steatorrhea.

Objective: The objective was to investigate whether portal FA absorption occurs in humans with cirrhosis.

Design: Six control subjects and 10 patients with (n = 5) and without (n = 5) cirrhotic ascites were fed [1-13C]palmitic and oleic acids in a test meal. Samples were drawn before and 30, 60, 90, 120, 240, 360, 480, and 720 min afterward for plasma [1-13C]-labeled FAs and breath 13CO2 assay. Fecal [1-13C]-labeled FAs were also measured.

Results: [1-13C]-Labeled FAs increased in chylomicrons in all groups, but less in ascitic cirrhotic patients, because their median area under the curve from 120 to 720 min was significantly lower than in the control subjects for labeled palmitate [520 (interquartile range: 192–1137) compared with 2862 (2674–4175) µmol · min/L] and oleate [829 (781–1263) compared with 3119 (2939–4986) µmol · min/L]. [1-13C]-Labeled FA enrichment of VLDL was also lower in cirrhotic patients. [1-13C]-Labeled FA in free FAs peaked earlier in ascitic than in nonascitic patients and control subjects, mainly for [1-13C]oleate, and the median area under the curve from 0 to 120 min was significantly higher in ascitic patients than in control subjects [301 (255–400) compared with 48 (34–185) µmol · min/L]. Fecal excretion of [1-13C]-labeled FA was negligible and not significantly different between groups.

Conclusions: The low [1-13C]-labeled FA concentrations in chylomicrons and VLDL, without increased fecal losses, confirm previous data in cirrhotic patients with the use of an unlabeled fat load. The earlier [1-13C]-labeled FA appearance in free FAs supports the portal absorption of dietary fat in patients with advanced cirrhosis with spontaneous portal-systemic shunting.

Key Words: Cirrhosis • fat absorption • stable isotopes • [1-13C]palmitic acid • [1-13C]oleic acid • lipoproteins • free fatty acids • mass spectrometry


INTRODUCTION  
About 40% of the adult energy requirement is supplied by fat, mostly as long-chain triacylglycerols. In the intestinal lumen, long-chain fatty acids (FAs) are hydrolyzed from triacylglycerols by pancreatic lipase, reesterified into mucosal triacylglycerols, and finally discharged as chylomicrons into the lymphatic vessels before reaching the thoracic duct and the systemic circulation (1). In contrast, short- and medium-chain FAs pass unesterified into the portal vein (2).

However, as early as the 1950s, studies in rats with biliary fistulas suggested that long-chain FAs do not always exit from gut in the lymph (3). Subsequently, studies in healthy rodents showed that between 30% and 70% of the intraduodenally infused long-chain FAs bypass the lymph and directly enter the portal vein (4, 5).

Whether portal absorption of long-chain FAs occurs in humans is unknown, but it could conceivably be of relevance in the setting of bile deficiency (6, 7). Intraluminal bile acid deficiency occurs in liver cirrhosis (8) as well as in other abnormalities, such as splanchnic lymphatic hypertension and intestinal lymphangiectasia (9–11), which may lead to impaired micelle formation and long-chain FA absorption through the usual lymphatic route. In fact, a blunted chylomicronemic response after an oral lipid load has been reported in cirrhotic persons (12, 13). However, because this occurred in the absence of significant steatorrhea (12, 13), an alternate route of absorption through the portal vein can be postulated. If the portal absorption of long-chain FAs occurs in cirrhosis, it could be of pathophysiologic importance because substantial amounts of fat could reach the liver in these patients.

To examine this hypothesis we conducted a study in cirrhotic patients and healthy volunteers to assess the incorporation of orally fed [1-13C]-labeled saturated (ie, palmitic acid, 16:0) and monounsaturated (ie, oleic acid, 18:1n-9) FAs into plasma lipids. Measurements of fecal [1-13C]-labeled FAs and 13CO2 breath excretion were also performed.


SUBJECTS AND METHODS  
Subjects
Six healthy volunteers and 10 cirrhotic patients with (n = 5) and without (n = 5) ascites were included in the study. Their demographic, clinical, and laboratory features are summarized in Table 1. Healthy volunteers did not have acute or chronic illness and had not undergone gastrointestinal or hepatobiliary surgery in the past, and their routine laboratory values were within the normal range.


View this table:
TABLE 1. Characteristics of the subjects1

 
All subjects abstained from tobacco and alcohol within the 30 d before the study and were not treated with insulin, glucose- or lipid-lowering drugs, ß-blockers, or glucocorticosteroids during this time. Lactulose, lactitol, cholestyramine, or other bile salt–chelating agents were also not allowed within the 3 d before the study.

Specific exclusion criteria for cirrhotic patients included the biliary etiology of the cirrhosis, the presence of hepatocarcinoma, active infection, acute gastrointestinal bleeding, overt hepatic encephalopathy, portal thrombosis (as assessed by ultrasonography), surgical or intrahepatic portal-systemic anastomosis, acute or chronic pancreatic disease, extrahepatic cholestasis, gastrointestinal surgery, dyslipemia, and fasting glycemia (glucose concentration > 7.7 mmol/L, or 140 mg/dL).

Study design
The study design is depicted in Figure 1. After a 12-h overnight fast, subjects were fed a mixture of [1-13C]-labeled FAs (1 g [1-13C]palmitic acid + 1 g [1-13C]oleic acid per person) (Isomed, Madrid) in 10 g sunflower oil with a standard meal consisting of 20 g margarine, 3 toasted bread slices, and 250 mL of a liquid nutritional supplement (Meritene; Novartis Consumer Health, SA, Barcelona, Spain). Subjects remained awake and in a semirecumbent position for the next 720 min. Venous blood samples for plasma [1-13C]-labeled FA assay were obtained at baseline and 30, 60, 90, 120, 240, 360, 480, and 720 min after tracer administration. The plasma was immediately separated by centrifugation and stored at –80°C until assayed. Also, end-expiratory breath samples for 13CO2 assay were obtained at these same time points and stored in sealed tubes. Fasting venous blood samples for plasma lipoprotein lipase (LPL), hepatic triacylglycerol lipase (HTGL), and lecithin-cholesterol acyltransferase (LCAT) assay were drawn in the morning of the next day. Finally, the total amount of feces yielded for 3 d after tracer administration was collected. Because the subjects were not allowed to eat during the 12-h study period, intravenous glucose (1 mg · kg body wt–1 · min–1) was infused to minimize FA mobilization from adipose tissue. This dose was empirically chosen on the basis of general recommendations for parenteral nutrition. With this regimen, there were no differences in the glycemic response between the 3 groups studied. In all subjects, glycemia peaked 60 min after the test meal. From 120 to 720 min, serum glucose remained between 5.5 and 8.5 mmol/L in all subjects.


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FIGURE 1.. Study design. FAs, fatty acids (ie, palmitic and oleic acids).

 
The study was performed in accordance with the latest revision of the Helsinki Declaration of 1975, as revised in 1983, and was approved by the Ethics Committee of the hospital. Informed consent was obtained from both the patients and the control subjects.

[1-13C]-Labeled fatty acid assay in plasma samples
Chylomicrons, VLDL, LDL, and HDL were isolated from plasma by sequential ultracentrifugation according to the Havel method (14). Heptadecanoic acid (15 µL of a 1 g/L solution) was added to 400 µL plasma as internal standard for free FA (FFA). On the other hand, known amounts of 10 g/L solutions of glyceryl triheptadecanoate, phosphatidyl cholyl heptadecanoate and cholesteryl heptadecanoate were added to each lipoprotein fraction, as internal standards for triacylglycerols, phospholipids, and cholesteryl esters, respectively. Assay of the [1-13C]-labeled FAs was performed by gas chromatography–mass spectrometry (GC-MS) as described in detail elsewhere (15)

Lipid extraction was performed in chloroform:methanol according to Bligh and Dyer (16). Then, FFAs, phospholipids, triacylglycerols, and cholesteryl esters were separated by thin-layer chromatography in silica gel plates with hexane:diisopropyl ether:acetic acid (80:20:1, by vol) as mobile phase. Lipid fractions were recovered from plates and reextracted with chloroform:methanol (2:1, by vol). The chloroform layer was dried and hydrolyzed to FAs in alcoholic potassium hydroxide at 70°C for 2 h. FAs were derivatized to trimethylsilyl (TMS) esters with bis-N,O-trimethylsilyl trifluoroacetamide + 5% TMS for 30 min at 70°C. TMS esters were then dried and redissolved in 1 mL hexane.

A volume of 1 µL of this extract was injected with the use of an autosampler AS 2000 (Thermo Quest, Milan, Italy) into a quadrupolar mass spectrometer MD 800 (Thermo Quest, Manchester, United Kingdom) operating in positive electronic impact set to100 µA, connected to a gas chromatograph CG 8060 (Thermo Quest, Milan, Italy) equipped with a J&W DB-1 (60m, 0.25 mm, 0.25 µm) column (Cromlab SA, Barcelona, Spain), and with helium as a carrier. Injection was performed on splitless mode at 300°C. TMS esters were separated at constant pressure (175 kPa) with the following temperature program: 165°C for 1 min, increase at a rate of 10°C/min up to 210°C, isotherm at 210°C for 27 min, a further increase at a rate of 40°C/min up to 315°C, and isotherm at 315°C for 5 min.

To assess both the isotopic enrichment and the individual FA concentrations (both natural and [1-13C]-labeled) with the same GC-MS run, a dual-acquisition program was designed in single-ion monitoring mode. This program records 2 signals (m and m + 1) from each FA. The following mass-to-charge ratios were acquired: 313.25 and 314.25 for TMS-palmitate, 327.27 and 328.27 for TMS-heptadecanoate, and 339.27 and 340.27 for TMS-oleate

Standard mixtures of labeled and unlabeled FAs were prepared gravimetrically to obtain different molar percent excesses (MPE) of labeled over unlabeled compounds. Aliquots of these standard mixtures were treated and derivatized as described above, and their MPE measured by GC-MS

Five calibration curves for isotopic enrichment of each FA were made by plotting the GC-MS obtained against the real gravimetrically obtained MPE values. Each curve was obtained by 50% dilution of the preceding one. The best calibration curve for deriving the FA concentration in plasma samples was selected in terms of the internal standard peak area that better fit with that of the sample. The concentration of each [1-13C]-labeled FA was finally calculated by multiplying its MPE by the concentration of its corresponding natural compound.

With this method, the isotopic ratio is not dependent on the amount of the analyte in the sample within a 10-fold range of concentration, with a maximum uncertainty of 0.34% in terms of MPE. In addition, both the within-day and between-day imprecision of the method were <1% (15). With this method, the recovery rates for both [1-13C]palmitic acid and [1-13C]oleic acid were 92% and 99%, respectively (15).

Breath 13CO2 assay
The breath 13CO2 assay was performed in duplicate by means of isotope ratio MS with an Automated Breath Carbon Analyzer Spectrometer (PDZ Europa, Cheshire, United Kingdom). 13C isotopic enrichment is expressed as by comparison with a 13C PDB international standard.

Assessment of total fat and [1-13C]-labeled fatty acids in feces
Three-day fecal samples were homogenized and diluted in acidified water (1:1, wt:vol); the lipids were hydrolyzed to FAs with potassium hydroxide, neutralized with hydrochloric acid, and extracted with 30 mL ether petroleum. Two aliquots of the extract were used for the total fecal fat (25 mL) and [1-13C]-labeled FA assay (5 mL). Total fecal fat was measured according to the method of van de Kamer et al (17). Fecal [1-13C]-labeled FAs were assayed with the same CG-MS method as described for plasma samples, with the addition of 1 mL of the heptadecanoic acid solution as internal standard.

Measurement of plasma LPL, HTGL, and LCAT activities
LPL and HTGL were measured in plasma obtained 10 min after intravenous sodium heparin (20 IU/kg body wt) stimulation as described previously (18). Plasma LCAT activity was measured with the method described by Stokke and Norum (19).

Statistical analysis
Data are presented as medians and interquartile ranges. For the time course of [1-13C]-labeled FAs and breath 13CO2, the area under the curve (AUC) was calculated by using the trapezoid rule and was split into 2 time periods: the AUC from baseline to 120 min (AUC0–120 min) and that from 120 to 720 min (AUC120–720 min). All AUCs were computed as above the minimum value.

Variations of labeled and unlabeled FAs and 13CO2 with time in either group were assessed by using Friedman’s within-group repeated-measures analysis of variance (ANOVA). Comparisons among groups at each time point were made by means of the Kruskal-Wallis ANOVA. Post hoc Mann-Whitney U tests were performed (and their P values adjusted for multiple comparisons with Bonferroni’s correction) only if significant differences or a significant interaction between time and group effects were found. The same methods were used to compare the AUCs among groups. Baseline comparison of qualitative variables (sex and etiology of cirrhosis) was made with a chi-square test. Statistical analysis was performed by using STATISTICA 5.5 software (StatSoft Inc, Tulsa OK).


RESULTS  
Time course of plasma [1-13C]-labeled fatty acid concentrations
In the 3 groups of subjects, both [1-13C]-labeled FAs increased in chylomicron-associated triacylglycerols early after the test meal, reached a plateau, and finally decreased to near baseline values. However, this response was blunted and less sustained in the cirrhotic patients than in the healthy control subjects, mainly in late samples (Figure 2, A and B; upper portions). This was confirmed by a significantly lower AUC120–720 min for [1-13C]palmitic acid (Figure 2A, lower right portion) and for [1-13C]oleic acid (Figure 2B, lower right portion) in chylomicrons in the ascitic cirrhotic patients than in the control subjects.


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FIGURE 2.. Time course and early (0–120 min) and late (120–720 min) areas under the curve (AUCs) for [1-13C]palmitic acid (A) and [1-13C]oleic acid (B) concentrations in chylomicron-associated plasma triacylglycerols from healthy control subjects and nonascitic and asctic cirrhotic patients. *Significantly different from the healthy subjects, P < 0.05 (Kruskal-Wallis ANOVA followed by a post hoc Mann-Whitney U test, adjusted for multiple comparisons with Bonferroni’s correction). The change with time was significant (P < 0.004) in every group for both fatty acids (Friedman’s within-group repeated-measures ANOVA). There were no significant time-by-treatment interactions. Data are expressed as medians; bars represent interquartile ranges.

 
Similarly, a decreased enrichment of VLDL-associated triacylglycerols with [1-13C]-labeled FAs, particularly [1-13C]oleic acid, was observed in cirrhotic patients (Figure 3, A and B, upper portions). This was also more apparent in the late samples, as evidenced by a lower AUC120–720 min for [1-13C]oleic acid in both ascitic and nonascitic patients than in the control subjects (Figure 3B, lower right portion).


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FIGURE 3.. Time course and early (0–120 min) and late (120–720 min) areas under the curve (AUCs) for [1-13C]palmitic acid (A) and [1-13C]oleic acid (B) concentrations in VLDL-associated plasma triacylglycerols from healthy control subjects and nonascitic and asctic cirrhotic patients. *Significantly different from the healthy subjects, P < 0.05 (Kruskal-Wallis ANOVA followed by a post hoc Mann-Whitney U test, adjusted for multiple comparisons with Bonferroni’s correction). The change with time was significant (P < 0.03) in every group for both fatty acids (Friedman’s within-group repeated-measures ANOVA). There were no significant time-by-treatment interactions. Data are expressed as medians; bars represent interquartile ranges.

 
The enrichment of FFAs with [1-13C]-labeled substrates peaked earlier in ascitic (at 120 min) than in nonascitic (at 240 min) cirrhotic patients than in healthy control subjects (at 360 min) (Figure 4, A and B, upper portions). Interestingly, these differences were particularly evident for [1-13C]oleic acid, as evidenced by its significantly higher concentration at 30 and 60 min (Figure 4B, upper portion) and AUC0–120 min in ascitic patients (Figure 4B, lower left portion).


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FIGURE 4.. Time course and early (0–120 min) and late (120–720 min) areas under the curve (AUCs) for [1-13C]palmitic acid (A) and [1-13C]oleic acid (B) concentrations in plasma free fatty acids from healthy control subjects and nonascitic and ascitic cirrhotic patients. *Significantly different from healthy subjects, P < 0.05 (Kruskal-Wallis ANOVA followed by a post hoc Mann-Whitney U test, adjusted for multiple comparisons with Bonferroni’s correction). #Significantly different from nonascitic patients, P < 0.05 (Kruskal-Wallis ANOVA followed by a post hoc Mann-Whitney U test, adjusted for multiple comparisons with Bonferroni’s correction). The change with time was significant (P < 0.02) in every group for both fatty acids (Friedman’s within-group repeated-measures ANOVA). There was a significant time-by-treatment interaction (P < 0.05) for [1-13C]oleic acid. Data are expressed as medians; bars represent interquartile ranges.

 
There were no significant differences in the time course of [1-13C]-labeled FA enrichment in the triacylglycerols of LDL and HDL between the 3 groups studied. Likewise, no detectable amounts of [1-13C]-labeled FA were found in the phospholipids and cholesteryl esters associated with the different plasma lipoproteins, both in the cirrhotic patients and the healthy control subjects (data not shown).

Time course of plasma total fatty acid concentrations
As expected, total (ie, unlabeled + [1-13C]-labeled) concentrations of both FAs increased after the test meal in the chylomicron-associated triacylglycerols of both the cirrhotic patients and the control subjects (Figure 5). However, there were no significant differences in the time course of FAs between groups (Figure 5, upper right and left portions).


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FIGURE 5.. Time course of total palmitic acid and oleic acid concentrations in chylomicron (Chm)- and VLDL-associated triacylglycerols (TG) and in plasma free fatty acids (FFAs) from healthy control subjects and nonascitic and ascitic cirrhotic patients. For both fatty acids, the change with time was significant (P < 0.008) in every group for Chm and FFAs, but not for VLDL (Friedman’s within-group repeated-measures ANOVA). There were no significant time-by-treatment interactions. Data are expressed as medians; bars represent interquartile ranges.

 
Little change in the total concentration of palmitic and oleic acids in the VLDL-associated triacylglycerols was observed in either group. However, total concentrations of both FAs (particularly oleic acid) remained lower in the ascitic patients than in the nonascitic cirrhotic patients and the healthy control subjects (Figure 5, middle panels).

Plasma concentrations of total palmitic and oleic acids in the FFA fraction were somewhat high at baseline, initially decreased after the test meal (reaching a nadir between 60 and 120 min), and recovered afterward. There were no significant differences in their time course between the 3 groups of subjects studied (Figure 5, lower panels).

Plasma total concentration of both FAs in the LDL- and HDL-associated triacylglycerols remained stable over time, and there were no significant differences between groups (data not shown).

Fecal excretion of total fat and [1-13C]-labeled fatty acids
No significant differences in the 3-d fecal excretion of either total fat or [1-13C]FA were found, among the 3 groups studied (Table 2).


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TABLE 2. Three-day fecal excretion of total fat and [1-13C]-labeled fatty acids1

 
Time course of breath 13CO2 excretion
Breath 13CO2 excretion showed a progressive increase during the study period in all groups. However, this was somewhat lower in the later samples (and therefore the AUC120–720 min) of ascitic patients (Table 3).


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TABLE 3. Time course and areas under the curve (AUCs) of breath 13CO2 excretion

 
Plasma activities of LPL, HTGL, and LCAT
As expected the plasma activities of both HTGL and LCAT were significantly lower in the nonascitic and ascitic cirrhotic patients than in the healthy control subjects. LPL activity was lower in the ascitic patients than in the healthy control subjects (Table 4).


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TABLE 4. Plasma activities of lipoprotein lipase (LPL), hepatic-triacylglycerol lipase (HTGL), and lecithin-cholesterol acyltransferase (LCAT)1

 

DISCUSSION  
The present study was designed to assess the absorption pathway and early destiny of dietary long-chain FAs in cirrhosis by tracing orally fed [1-13C]-labeled palmitic and oleic acids—the most abundant dietary FAs—in plasma lipoproteins and feces of cirrhotic patients and healthy control subjects. Because the number of subjects in the study was relatively small, the severity of cirrhosis was defined by the single criterion of the presence of ascites (rather than the widely used Child-Pugh composite index), to render the cirrhotic groups more homogeneous. Also, because of the variable incidence and severity associated with exocrine pancreatic insufficiency in cirrhosis (20), we administered the labeled substrates as unesterified FAs instead of as triacylglycerols.

The incorporation of [1-13C]-labeled FAs in chylomicron- and VLDL-associated plasma triacylglycerols was lower and less sustained in cirrhotic patients than in healthy control subjects, whereas their fecal excretion was similar in both groups. The lower incorporation of labeled FAs in VLDLs is consistent with the well-known impaired export of these lipoproteins from the cirrhotic liver (21, 22), probably because of a decreased synthesis of triacylglycerols (23) and apolipoprotein E (24) in the liver of these patients. Likewise, the decreased enrichment of chylomicrons with labeled substrates confirms the findings of previous studies, which reported a low chylomicronemic response in cirrhotic patients after an oral load of unlabeled fat (12, 13). Indeed, the impaired incorporation of dietary fat (either labeled or unlabeled) in chylomicrons, in the absence of significant fecal losses, strongly supports an alternate absorption route of fat in cirrhosis.

In this sense, the finding that the incorporation of [1-13C]-labeled FAs into plasma FFAs peaked much earlier in the ascitic cirrhotic patients than in the healthy control subjects (with intermediate values for nonascitic cirrhotic patients) is revealing. In fact, an increase in plasma FFAs after an oral fat load in cirrhotic patients was previously reported (25), although it was masked in part by the increased fasting FFA concentrations in these patients (26). Thus, the use of labeled substrates in the present study was particularly useful to confirm that the postprandial increase in plasma FFAs in cirrhosis is, at least in part, of dietary origin. In our view, the best explanation for this phenomenon is that a certain proportion of labeled FAs are absorbed through the portal vein and partly enter the systemic circulation, bypassing the liver, through the portal-systemic collateral vessels present in advanced cirrhotic patients with portal hypertension. An alternative explanation could be that [1-13C]-labeled FAs may have been released by the action of LPL on triacylglycerols of chylomicrons. Indeed, recent studies in healthy subjects fed [1-13C]-labeled FAs indicate that the release of FAs hydrolyzed from triacylglycerols escaping entrapment in adipose tissue mostly account for the enrichment of plasma FFAs with [1-13C]-labeled FAs (27). As in our control group, this does not occur before 120 min and peaks at 360 min (27). Thus, it seems unlikely that the early increase (before 120 min) in [1-13C]-labeled FAs in plasma FFAs occurring in our ascitic cirrhotic patients was due to this mechanism. Moreover, the low plasma LPL activity in these patients strongly argues against this possibility.

The possibility that, in certain pathologic conditions, dietary long-chain FAs could be absorbed via pathways not dependent on their incorporation into chylomicrons and subsequent transport through the lymphatic system was already hypothesized by the end of the 1960s. In a study conducted in patients with complete biliary obstruction, in whom the thoracic duct was cannulated, Blomstrand et al (6) showed that feeding [14C]-labeled FAs resulted in low amounts of radioactivity in the lymph from the thoracic duct, together with high radioactivity in feces, but also in the expired air, which suggests the existence of some alternative means of long-chain FA absorption in the absence of bile in the intestinal lumen (6). Similar conclusions were drawn from studies in patients with external biliary drainage in whom the consumption of fat-soluble vitamins increased their plasma concentrations in the absence of both significant steatorrhea and a chylomicronemic response (7).

Indeed, portal absorption of dietary FAs occurs in healthy rats, to an extent ranging from 30% to 70%, depending on the intraduodenally infused FA (4, 5). The portal route of absorption seemed to be particularly efficient for unsaturated rather than for saturated FAs (4), as occurred in our ascitic patients in whom the early incorporation to plasma FFAs was more evident for [1-13C]oleic (ie, unsaturated) acid than for [1-13C]palmitic (ie, saturated) acid.

The fact that significant amounts of dietary fat could be absorbed through the portal route in cirrhosis has both pathophysiologic and therapeutic implications. It is conceivable that the inflow of fat into the liver via the portal vein, combined with the abovementioned impairment in VLDL release by the liver, could facilitate the storage of fat in the liver of cirrhotic patients. In fact, a long-term increase in plasma FAs of dietary origin (namely saturated, monounsaturated, and essential FAs)—but not of those mainly depending on hepatic synthesis (ie, long-chain polyunsaturated FAs)—was reported in cirrhotic patients undergoing portacaval anastomosis compared with unoperated control subjects (28). Because the nutritional status improved similarly in both groups (28), it might well be that the increase in plasma FAs after portacaval anastomosis was due to an increased systemic availability of portally absorbed FAs no longer sequestered in the liver. If this phenomenon is true, fat accumulation could be an additional contributing factor to hepatic inflammation and fibrogenesis in these patients (29), and spontaneous portal-systemic shunting should be viewed as a protective mechanism. Whether the portal absorption of dietary fat has any role in accelerating the progression of chronic liver disease is an issue to be addressed in future investigations.

In the meantime, however, this possibility has to be kept in mind when recommending a diet for cirrhotic patients, particularly in the design of formulas for enteral tube feeding. In rats, the portal absorption of FAs is proportionally greater when low intraduodenal infusion rates are used (30). Accordingly, it may also be particularly efficient in cirrhotic patients receiving continuous low-rate enteral tube feeding. In our experience, cirrhotic patients present a virtually absent chylomicronemic response while receiving lipid-containing continuous enteral nutrition (31). This finding suggests that, under these circumstances, portal lipid absorption is maximal. In this setting, a low-fat diet with a fat source mainly consisting of saturated FAs should, in theory, be preferred. In fact, saturated FAs have been reported to protect against hepatic steatosis in animal models of alcoholic liver disease (32–34).

In summary, cirrhotic patients have a lower incorporation of [1-13C]-labeled FAs in chylomicrons and VLDL, in the absence of increased fecal losses, which confirms the previous data from studies that used an unlabeled fat load. The earlier appearance of [1-13C]-labeled FAs in plasma FFAs favors the portal absorption of (mainly unsaturated) long-chain FAs in advanced cirrhosis with portal hypertension and portal-systemic collaterals. Whether portal FA absorption could be a risk factor for hepatic steatosis in these patients deserves further research.


ACKNOWLEDGMENTS  
EC designed the experiment, selected the patients, collected the samples, performed the statistical analysis, and wrote the manuscript. JMH-P analyzed the different samples and contributed to the statistical analysis and the writing of the manuscript. LF helped analyze the samples. CP and AC provided significant advice and consultation regarding the laboratory procedures. MAG contributed to the design of the experiment and the critical review of the manuscript. None of the authors declared a conflict of interest


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Received for publication July 12, 2004. Accepted for publication November 8, 2004.


日期:2008年12月28日 - 来自[2005年81卷第3期]栏目

Resting energy expenditure in diabetic and nondiabetic patients with liver cirrhosis: relation with insulin sensitivity and effect of liver transplantation an

Gianluca Perseghin, Vincenzo Mazzaferro, Stefano Benedini, Andrea Pulvirenti, Jorgelina Coppa, Enrico Regalia and Livio Luzi

1 From the Division of Internal Medicine, Section of Nutrition/Metabolism (GP, SB, and LL) and the Unit of Clinical Spectroscopy (GP and LL), Istituto Scientifico H San Raffaele, Milan, Italy, and the Hepato-Pancreatic Surgery and Liver Transplantation Unit, National Cancer Institute, Milan, Italy (VM, AP, JC, and ER).

2 Supported by Istituto Scientifico H San Raffaele (PZ709 and PZ806) and Telethon-Italy (1032C). G Perseghin received grants from the Italian Ministry of Health (RF96.305, RF98.49, and RF99.55) and the Italian National Research Council (CNR 97.00485.CT04). L Luzi (Protein metabolism in patients with hepatocarcinoma before and after liver transplantation) and V Mazzaferro (New therapeutic strategies for hepatocellular carcinomas) received grants from the Associazione Italiana Ricerca Cancro.

3 Address reprint requests to G Perseghin, Division of Internal Medicine, Unit of Clinical Spectroscopy, Section of Nutrition/Metabolism via Olgettina 60, 20132 Milan, Italy. E-mail: perseghin.gianluca{at}hsr.it.


ABSTRACT  
Background: Hypermetabolism, insulin resistance, and diabetes are common in patients with liver cirrhosis.

Objective: We assessed whether diabetes and insulin resistance influence postabsorptive energy homeostasis in these patients and whether liver transplantation (LTx) and immunosuppressive drugs affect these relations.

Design: Twenty-six patients with liver cirrhosis (16 with and 10 without diabetes) were studied with an insulin clamp and indirect calorimetry. Eleven of these subjects were studied 9 mo after LTx to longitudinally assess its effects. To cross-sectionally explore a longer follow-up period, we studied 65 patients 6, 14, and 32 mo after LTx. Seven patients with chronic uveitis (receiving immunosuppressive therapy) and 20 healthy subjects served as control subjects.

Results: Diabetic and nondiabetic patients with cirrhosis had insulin resistance (SI(clamp); P < 0.03) and higher measured resting energy expenditure (REE) as a percentage of predicted REE than did healthy subjects (107.6 ± 1.8% compared with 97.4 ± 2.3%; P < 0.03), and these 2 alterations were associated (R2 = 0.119, P = 0.0002). The longitudinal study showed an improvement in the 2 variables after LTx, but full restoration was not achieved. The cross-sectional analysis confirmed this observation in patients studied 6 mo (n = 28) after LTx. In patients studied 14 (n = 21) and 32 mo (n = 16) after LTx, SI(clamp) and measured REE as a percentage of predicted REE were not significantly different from those in control subjects.

Conclusions: In patients with liver cirrhosis, higher-than-normal postabsorptive REE was associated with insulin resistance regardless of diabetes. This abnormality persisted in patients studied 6–9 mo after LTx but improved simultaneously with the improvement in insulin sensitivity thereafter.

Key Words: Resting energy expenditure • liver cirrhosis • insulin resistance • liver transplantation • lipid oxidation • immunosuppressive therapy • diabetes


INTRODUCTION  
Hypermetabolism may occur in patients with liver cirrhosis regardless of the clinical, laboratory, or histologic features of the disease or of its duration and severity (1), with the exception of primary biliary cirrhosis (2), suggesting that extrahepatic factors are the major determinants of hypermetabolism. Identification of these factors is therefore important because hypermetabolism, which induces malnutrition, may have a profound impact on the prognosis of patients with liver cirrhosis (1). Because patients with extrahepatic portal obstruction (3) or liver transplants (4) are hypermetabolic (despite having a normal liver function), portal hypertension and portosystemic shunting are suggested to be causative factors. In addition, the effects of the systemic inflammatory condition, which is mediated by increased concentrations of various cytokines (5), and the effects of an increased ß-adrenergic activity were suggested to cause hypermetabolism in clinically stable patients with liver cirrhosis (1).

Insulin resistance is highly prevalent in patients with liver cirrhosis (6), is a primary event in the development of hepatogenous diabetes (7), and also influences the pathology and natural history of hepatogenous diabetes (8). The fact that the insulin-mediated oxidative disposal of carbohydrates (6, 9), lipids (9), and proteins (10) in liver cirrhosis is altered in association with increased ß-adrenergic activity induced us to hypothesize that hypermetabolism in cirrhosis may be mediated by insulin resistance and overt diabetes.

This study was therefore undertaken to test whether postabsorptive energy homeostasis in patients with liver cirrhosis is related to insulin resistance and is made worse by diabetes and to test whether liver transplantation (LTx), which improves insulin sensitivity (11, 12), might also influence resting energy expenditure (REE) and substrate oxidation rates despite the administration of immunosuppressive drugs. We pursued this aim with a longitudinal approach involving 11 cirrhotic patients who were studied before and 9 mo after LTx and with a cross-sectional approach involving 26 cirrhotic patients and 65 patients with liver transplants who were studied 6, 14, and 32 mo after LTx. The cross-sectional study was designed to explore a longer follow-up period after the surgical procedure and included a group of patients with a nonsystemic disease (chronic uveitis) who were treated with an immunosuppressive regimen similar to that of transplant patients.


SUBJECTS AND METHODS  
Subjects
Longitudinal study
Eleven patients with postnecrotic cirrhosis (Child-Pugh class A or B) complicated by small unresectable hepatocellular carcinoma (for a single tumor, a tumor diameter < 5 cm; for multiple tumors, a tumor diameter < 3 cm) were recruited in the Hepato-Pancreatic Surgery and Liver Transplantation Unit of the National Cancer Institute. Five of these patients were also diagnosed as having diabetes (3 separate fasting plasma glucose values > 7 mmol/L), which was treated with diet or daily subcutaneous insulin injections. None had metastatic disease at the time of transplantation or when this study was performed. They were studied twice (before and 9 mo after LTx) by means of the euglycemic-hyperinsulinemic clamp and indirect calorimetry as described below. After transplantation, all patients were in stable clinical condition; liver function was normal, with the exception of slight increases in alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1). Two of the 5 patients with diabetes had normal fasting glucose concentrations after LTx; therefore, at the time the study was repeated, 3 of the 11 patients were still diabetic (2 were being treated with diet and 1 with insulin). The anthropometric, laboratory, and clinical characteristics of patients and control subjects [7 patients with chronic uveitis (CU) who were receiving immunosuppressive therapy similar to that of transplant patients and 20 healthy subjects] in this study are shown in Table 1.


View this table:
TABLE 1 . Longitudinal study: anthropometric, laboratory, and clinical characteristics of cirrhotic patients before and 9 mo after liver transplantation (LTx) and of control subjects1  
Cross-sectional study
To explore a longer follow-up period, we performed a cross-sectional study that included 1) 26 cirrhotic patients (16 with diabetes) who had features similar to those of the patients enrolled in the longitudinal study, 2) 28 patients (7 with diabetes) who were studied 6 mo after LTx, 3) 21 patients (2 with diabetes) who were studied 14 mo after LTx, and 4) 16 patients (1 with diabetes) who were studied 32 mo after LTx. The eleven patients who participated in the longitudinal study were also included in the cross-sectional analysis: all 11 were included in the group of cirrhotic patients before LTx, 9 of them were included in the subgroup of patients who were studied 6 mo after LTx, and 2 of them were included in the subgroup of patients who were studied 14 mo after LTx. The anthropometric, laboratory, and clinical characteristics of these study groups are shown in Table 2.


View this table:
TABLE 2 . Cross-sectional study: anthropometric, laboratory, and clinical characteristics of cirrhotic patients before liver transplantation (LTx) and of LTx patients who were studied 6, 14, and 32 mo after LTx 1  
The potential effect of diabetes on REE before LTx was also analyzed in the 26 patients with liver cirrhosis by comparing those with diabetes (n = 16) with those without (n = 10). All subjects who underwent the experimental procedures were fully informed of the possible risk of the study and gave their consent. The experimental protocol was approved by the Ethical Committee of the Istituto Scientifico H San Raffaele.

Experimental protocol
Subjects were instructed to consume an isoenergetic diet and to abstain from exercise activity for 3 wk before the studies. At the time of the studies, patients with diabetes were being treated with diet or insulin and were not receiving oral hypoglycemic agents. The use of drugs with potential metabolic effects was discontinued for a washout period of 3 d, and patients with diabetes were instructed to receive the last doses of intermediate- and short-acting insulin 24 and 12 h, respectively, before the study. Administration of immunosuppressive drugs was withheld on the morning of the study. At 0700 on the fourth day, subjects were admitted to the Metabolic Unit of the Division of Internal Medicine I of the Istituto Scientifico H San Raffaele after a 10-h overnight fast, and the following procedures were performed.

Indirect calorimetry
After subjects had lain quietly for 30 min, REE was measured for 45 min by continuous indirect calorimetry with a ventilated hood system (SensorMedics 2900 Metabolic Measurement Cart; SensorMedics, Milano, Italy) as previously described (7). The mean (± SE) CV within the session for both oxygen (1.6 ± 0.2%) and carbon dioxide (1.9 ± 0.3%) measurements were < 2%, with ranges of 0.3–4.8% and 0.4–5.6% for oxygen consumption (CO2. Hypermetabolism was defined as previously described ( Euglycemic-hyperinsulinemic clamp
When indirect calorimetry was performed, a polytetrafluoroethylene catheter was inserted into an antecubital vein for infusions, and an additional catheter was retrogradely inserted into a wrist vein for blood sampling. The subject’s hand was kept in a heated box (50 °C) throughout the experiment to allow sampling of arterialized venous blood. Blood samples for measurement of postabsorptive plasma glucose and free insulin were obtained in triplicate. Thereafter, a euglycemic-hyperinsulinemic clamp was performed as previously described (7, 13). Insulin was infused at 40 mU • m-2 • min-1) to reach a plasma insulin concentration of 400 pmol/L, and the plasma glucose concentration was kept at 5 mmol/L for 150 min by variable infusion of a 20% (by vol) dextrose solution. Blood samples for plasma insulin and glucose were drawn every 15 min throughout the study.

Analytic procedures
Plasma glucose was measured with a Beckman glucose analyzer (Fullerton, CA; 7), and the mean (± SE) CV was 1.7 ± 0.1% and 3.0 ± 0.4% in the fasting and clamp conditions, respectively. Plasma free insulin was measured as previously described (7), and the mean CV was 12.2 ± 1.7% and 5.7 ± 0.7% in the fasting and clamp conditions, respectively. The Kjeldahl method (14) was used to measure urine nitrogen in 24-h urine samples that the patients collected on the previous day.

Calculations
Indirect calorimetry
REE was calculated from the oxygen consumption and carbon dioxide production rates measured by means of indirect calorimetry (excluding the first 10 min of data acquisition) and from the urinary nitrogen excretion by using Weir’s standard equation (15). Predicted REE was calculated by using the Harris-Benedict equations (16). Glucose, lipid, and protein oxidation were estimated as previously described (17).

Insulin sensitivity
The steady-state glucose infusion rate (GIR) was measured during the 120–150 min of the insulin clamp and was expressed as mg • kg body wt-1 • min-1; the mean (± SE) CV was 4.9 ± 0.5%. The clamp-related index of insulin sensitivity (SI(clamp)) was calculated as GIR/(I x G), where I is the change in plasma insulin concentration during the last 30 min of the clamp from that of baseline and G is the plasma glucose concentration during the same interval (18).

Statistical analysis
All data are presented as means ± SEMs. With the use of standard linear regression, steady state was defined as a nonsignificant correlation of the variable with time (P > 0.05). Relations between variables were assessed by using linear regression analysis. In the longitudinal study of patients with liver cirrhosis, values before and after LTx were compared by using Student’s paired t test, and the P values were Bonferroni corrected to adjust for the fact that the means were also used for comparison with the means for CU patients and healthy subjects. Values for the groups in the cross-sectional study were compared by using one-way analysis of variance and Tukey’s post hoc test.

Measured REE as a percentage of predicted REE was stratified into 6 subgroups (I: 120%; II: 110% but < 120%; III: 100% but < 110%; IV: 90% but < 100%; V: 80% but < 90%; VI: < 80%) within the 26 patients with liver cirrhosis, within the 65 transplant patients (regardless of the age of the transplant), and within the 27 control subjects (CU patients and healthy subjects). Differences in anthropometric variables, SI(clamp), and basal glucose and lipid oxidation between the members of each of the 3 groups (ie, the patients with liver cirrhosis, the LTx patients, and the control subjects) and between all the study subjects pooled together in subgroups I, II, III, and IV were assessed by using one-way analysis of variance and Tukey’s test for post hoc comparisons. A trend effect of REE classes was also investigated by using the general linear models procedure. All analyses were performed with the use of SAS 6.12 (SAS Institute Inc, Cary, NC).


RESULTS  
Prevalence of hypermetabolism in study groups
In the patients with liver cirrhosis, the prevalence of hypermetabolism did not differ between those with (3 of 16; 19%) and without (2 of 10; 20%) diabetes. In the LTx patients studied 6, 14, and 32 mo after LTx, the prevalence of hypermetabolism was 7% (2 of 28), 9.5% (2 of 21), and 0%, respectively. No hypermetabolic individuals were found among the CU patients and healthy subjects. Two LTx patients were hypometabolic with normal thyroid function.

Effect of liver transplantation: longitudinal study
Anthropometric and laboratory characteristics of the study subjects
In comparison with healthy subjects matched for anthropometric characteristics, the cirrhotic patients before LTx were characterized by postabsorptive hyperglycemia (P < 0.01) and hyperinsulinemia (P < 0.01). After LTx, these patients had significant improvements in plasma glucose (P < 0.05) and insulin concentrations (P < 0.01) (Table 1).

Insulin sensitivity
The patients with liver cirrhosis had significantly lower values for GIR and SI(clamp) than did the healthy subjects (P < 0.03; Table 1), which reflected a marked insulin resistance with respect to glucose metabolism in the patients; this insulin resistance significantly improved 9 mo after LTx (P < 0.01) but was still significantly higher than that of the healthy subjects (P < 0.03).

Resting energy expenditure and substrate oxidation rates
The groups did not differ significantly in REE; nevertheless, measured REE values as a percentage of predicted REE (predicted on the basis of the classical Harris-Benedict equations) were slightly but significantly higher in the cirrhotic patients than in the healthy subjects (P < 0.03; Table 1). After LTx, REE was not completely normalized in comparison with that of healthy subjects (P = 0.05). In addition, either before or after LTx, patients with liver cirrhosis showed a trend for lower glucose oxidation (P = 0.08) with a parallel trend for higher lipid oxidation (P = 0.06).

Effect of liver transplantation: cross-sectional study
Anthropometric and laboratory characteristics of the study subjects
The study groups did not differ significantly in anthropometric variables (Table 2). The laboratory features of the patients with liver cirrhosis were comparable to those observed in the longitudinal study: in comparison with the healthy subjects, the cirrhotic patients had postabsorptive hyperglycemia (P < 0.03) and hyperinsulinemia (P < 0.03). In the patients who were studied 6 mo after LTx, fasting plasma glucose concentrations were not significantly different from those of the control subjects (P = 0.07) even though 7 of the patients still had diabetes. The plasma free insulin concentrations of the patients who were studied 6 mo after LTx were significantly lower than those of the patients who were studied before LTx (P < 0.05) but were not significantly different from those of the healthy subjects. In patients studied 14 and 32 mo after LTx, plasma glucose and free insulin concentrations were not significantly different from those of the healthy subjects (P = 0.15 and 0.27, respectively).

Insulin sensitivity
The patients with liver cirrhosis had significantly lower values for GIR and SI(clamp) than did the healthy subjects (P < 0.03; Table 2), which reflected a marked insulin resistance in the patients. The patients who were studied 6 mo after LTx had SI(clamp) values that were significantly lower than those of the healthy subjects (P < 0.03). In the patients who were studied 14 or 32 mo after LTx, SI(clamp) was not significantly different from that of healthy subjects (P = 0.72 and 0.61, respectively). GIR values showed a parallel behavior.

Resting energy expenditure and substrate oxidation rates
The groups did not differ significantly in REE; nevertheless, measured REE values as a percentage of predicted REE were slightly but significantly higher in the cirrhotic patients than in the healthy subjects (P < 0.03; Table 2). In the patients who were studied 6 mo after LTx, measured REE as a percentage of predicted REE was not significantly different from that of the healthy subjects (P = 0.06) or that of the patients who were studied 14 (P = 0.287) or 32 (P = 0.08) mo after LTx. In association with these results, oxidative substrate disposal was not significantly different between the groups, even though there were nonsignificant trends for higher lipid oxidation (P = 0.07) and lower glucose oxidation (P = 0.08) in the patients with liver cirrhosis.

Effect of immunosuppressive drugs
The CU patients were not significantly different from the other study groups in terms of anthropometric variables (Table 1). The CU patients were characterized by normal fasting concentrations of plasma glucose but were slightly hyperinsulinemic in comparison with the healthy subjects (P = 0.05). GIR and SI(clamp) values were significantly lower in the CU patients than in the healthy subjects (P = 0.09 and 0.05, respectively). In the CU patients, the values for REE (P = 0.96), measured REE as a percentage of predicted REE (P = 0.47), respiratory quotient (P = 0.81), and glucose (P = 0.85) and lipid oxidation (P = 0.87) were not significantly different from those of the healthy subjects. The CU patients were taking prednisone and cyclosporin A at doses similar to those taken by the LTx patients who were studied 6 mo after LTx. The LTx patients who were studied 14 and 32 mo after LTx were receiving similar doses of cyclosporin A but no prednisone.

Effect of liver cirrhosis and diabetes on resting energy expenditure
Among the patients with liver cirrhosis, those with diabetes did not differ significantly from those without diabetes in anthropometric variables (Table 3). The patients with diabetes had significantly higher fasting plasma glucose concentrations than did those without diabetes (P < 0.01); fasting plasma free insulin concentrations did not differ significantly between the 2 groups (P = 0.58). When either the GIR (P = 0.09) or, more properly, the SI(clamp) (P = 0.11) was used as a variable of a clamp-derived index of insulin sensitivity, there was a nonsignificant trend for insulin sensitivity to be lower in the diabetic patients than in the nondiabetic patients (Table 3). REE was not significantly different between the 2 groups when expressed as MJ/d (P = 0.74), as kJ • kg body wt-1 • d-1 (P = 0.62), or as a percentage of the predicted value (P = 0.99). Basal glucose oxidation and lipid oxidation were also not significantly different between the 2 groups (P = 0.34 and 0.36, respectively; Table 3).


View this table:
TABLE 3 . Effect of diabetes in patients with liver cirrhosis: anthropometric, laboratory, and clinical characteristics1  
Stratification
To further detect the metabolic features of the study groups with respect to different REE rates, the anthropometric variables, SI(clamp), and glucose and lipid oxidation in the postabsorptive state were summarized in Table 4, in which the subjects are classified on the basis of measured REE as a percentage of predicted REE as hypermetabolic ( 120%, subgroup I), normometabolic (< 120% and 80%; subgroups II–V), and hypometabolic (< 80%). The different subgroups did not differ significantly in anthropometric variables. When the results of all 118 subjects in the cross-sectional study were pooled together and analyzed, there were significant trends for SI(clamp) to be lower (P < 0.003) and for lipid oxidation to be higher (P < 0.0001) in subjects with higher values of measured REE as a percentage of predicted REE. When the analysis was performed within each of the 3 main groups shown in Table 4, measured REE as a percentage of predicted REE was significantly associated with lipid oxidation (P < 0.05) in the LTx patients.


View this table:
TABLE 4 . Stratification of the study subjects on the basis of measured resting energy expenditure (REE) as a percentage of predicted REE1  
Regression analysis
To further test the hypothesis that hypermetabolism in liver cirrhosis was associated with insulin resistance, we plotted the data from all of the subjects who participated in the cross-sectional study and performed a simple regression analysis that showed that measured REE as a percentage of predicted REE (Figure 1) was inversely associated with SI(clamp) (R2 = 0.119, P = 0.0002).


View larger version (15K):
FIGURE 1. . Linear regression analysis of data from the cross-sectional study in which measured resting energy expenditure (REE) as a percentage of predicted REE is plotted against the clamp-related index of insulin sensitivity [SI(clamp)]. , patients with liver cirrhosis; •, liver transplantation (LTx) patients who were studied 6 mo after LTx; •, LTx patients who were studied 14 mo after LTx; , LTx patients who were studied 32 mo after LTx; , healthy subjects. R2 = 0.119, P = 0.0002.

 

DISCUSSION  
The present study is the first study in which postabsorptive energy homeostasis, substrate oxidation, and insulin sensitivity were simultaneously monitored in a longitudinal fashion in patients with liver cirrhosis before and after LTx; the study also includes the longest cross-sectional follow-up study (32 mo) of these variables. The results indicate that elevated REE in patients with liver cirrhosis is associated with reduced insulin action on glucose metabolism, suggesting that insulin resistance may play a role in the pathogenesis of hypermetabolism in cirrhotic patients. To sustain this conclusion, we found that the higher REE characterizing the pretransplant condition progressively decreased after LTx and was associated with a progressive improvement in insulin sensitivity and that measured REE as a percentage of predicted REE was inversely associated with SI(clamp) (Figure 1). In addition, when we compared the patients with liver cirrhosis on the basis of the presence or absence of diabetes, we also showed that diabetes per se was not associated with measured REE as a percentage of predicted REE.

The data presented in the present study confirm that liver cirrhosis is also characterized by higher-than-normal REE (1) in patients with a mild form of the disease or with hepatocarcinoma because all the patients in this study were classified as Child-Pugh class A or B. The prevalence of true hypermetabolism in this group of cirrhotic patients was 19% (Tables 2 and 4), which is probably not significantly different from the prevalence (28–29%) in the Child A cirrhotic patients with hepatocarcinoma described by Müller et al (1). Note that 91% of the cirrhotic patients had an REE higher than predicted, whereas this was true in only 38% of the control subjects (CU patients and healthy subjects), in whom true hypermetabolism was not found. In the 65 LTx patients, the prevalence of true hypermetabolism was 6%; yet, 52% of the patients had a measured REE higher than predicted (Table 4).

Substrate oxidation rates in the postabsorptive state were also affected. In fact, we noticed that in the 26 patients with liver cirrhosis, the postabsorptive lipid oxidation rate, on average, contributed 62% of the REE (data not shown), and this contribution was higher than in the healthy subjects (50%); this finding was associated with a lower contribution of carbohydrate oxidation in the cirrhotic patients than in the healthy subjects (22% compared with 34%; data not shown), as previously described in patients with more advanced stages of liver disease (19, 20). In addition, higher postabsorptive lipid oxidation rates were significantly associated with hypermetabolism when data from all of the subjects who participated in the cross-sectional study were pooled together (Table 4). Therefore, in agreement with the results of previous studies, higher lipid oxidation seemed to play a role in the development of hypermetabolism; nevertheless, in the cirrhotic patients with measured REE > 110% of predicted REE, hypermetabolism was also paralleled by normal-to-higher carbohydrate oxidation rates (Table 4), suggesting that in these patients the oxidative pathways are deeply affected at the level of both glucose and fatty acid oxidative disposal.

The pathogenesis of hypermetabolism in liver cirrhosis is still unknown, but several factors have been suggested to be involved: portal hypertension, systemic inflammation, and increased sympathetic nervous system activity (1, 3–5). The results of the present study suggest that insulin resistance may also be causative. In this study, insulin sensitivity with respect to glucose metabolism was measured by using the gold standard technique, the euglycemic-hyperinsulinemic clamp: the patients with liver cirrhosis had dramatically lower SI(clamp) values than did the healthy subjects, and these values were significantly associated with the severity of the hypermetabolic state (Figure 1). The observation that in the other human model of marked insulin resistance without liver injury, ie, obesity, hypermetabolism was not prevalent and insulin sensitivity was not associated with resting thermogenesis (21) would suggest that the relation between the hypermetabolic state and insulin resistance is peculiar to liver cirrhosis.

Further support for this view comes from the data obtained after LTx, which were analyzed by using both the longitudinal and cross-sectional approaches. The longitudinal study showed that 9 mo after LTx, the cirrhotic patients were still characterized by higher-than-normal measured REE/predicted REE (22), and this was paralleled by the persistence of insulin resistance with respect to glucose metabolism. The cross-sectional study showed that in the patients studied 14 or 32 mo after LTx, the complete restoration of insulin sensitivity was associated with measured REE/predicted REE values that were not significantly different from those in the healthy subjects.

Other metabolic and nonmetabolic variables may have a profound impact on REE. Development of hepatogenous diabetes is common in patients with liver cirrhosis. In fact, 45% of the patients who participated in the longitudinal study and 35% of those who participated in the cross-sectional study were diagnosed as having diabetes. In addition, LTx patients, especially within the first 12 mo after the transplant, showed some prevalence of diabetes (27% in the longitudinal study and 25%, 14%, and 3% in the patients who were studied 6, 14, and 32 mo, respectively, after LTx in the cross-sectional study). Because diabetes per se usually induces an insulin-resistant state (23), the presence of diabetes may further worsen the alteration in REE in cirrhotic patients; however, in the present study REE was not significantly higher in the diabetic patients than in the nondiabetic patients (Table 3), suggesting that diabetes per se is not involved in the hypermetabolic state and that insulin resistance represents the major adverse metabolic event associated with elevated REE.

The patients with liver cirrhosis were also affected by hepatocellular carcinoma, and for this reason they were eligible for LTx, which was previously shown to provide a better prognosis than that provided by liver resection (24). An independent cancer-related role in elevated REE is unlikely because no patients had disseminated disease at the time of the study and because patients with other chronic hepatic diseases without the associated development of hepatocarcinoma were shown to develop hypermetabolism as well. Nevertheless, this possibility may not be excluded.

Immunosuppressive drugs used to avoid graft rejection might also be involved in the alterations in REE in LTx padients, and this effect might also be dose dependent. We therefore selected and studied a group of CU patients who were undergoing an immunosuppressive regimen similar to that of LTx patients but who were without a systemic disease: the CU patients were selected to be similar to the study subjects in terms of drug therapy, anthropometric features, and lifestyle. We found that these patients did not differ significantly from the healthy subjects in terms of REE and fuel oxidative partitioning (Table 1). The combined administration of prednisone and cyclosporine A were associated only with a mild degree of insulin resistance, and prednisone in particular may be responsible for this defect. The absence of a significant effect on REE suggests that these drugs may have only a minor role, if any, on the persistence of higher than predicted REE in cirrhotic patients after LTx.

In conclusion, the results of this study show that hypermetabolism in liver cirrhosis complicated by hepatocarcinoma may be partly explained by insulin resistance and that the presence of diabetes does not play a major additional role in elevated REE. Using both the longitudinal and cross-sectional approaches, we found that the metabolic profile of LTx patients who were studied 32 mo after LTx progressively improved in terms of insulin action and postabsorptive REE despite the use of immunosuppressive drugs. The data suggest that monitoring of both energy homeostasis and insulin action in patients with liver cirrhosis before LTx and during the first months after LTx is mandatory for designing a program of nutritional and pharmacologic support to prevent malnutrition and metabolic complications in these patients.


ACKNOWLEDGMENTS  
We thank Antonella Scollo of the Metabolic Unit of the Istituto Scientifico H San Raffaele for nursing assistance, the Hepato-Pancreatic Surgery and Liver Transplantation Unit of the National Cancer Institute for excellent assistance, Giliola Calori of the Biometrical Unit of the Istituto Scientifico H San Raffaele for tremendous help with statistical analysis, and Cinzia Degani for editorial assistance.


REFERENCES  

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Received for publication July 10, 2001. Accepted for publication October 26, 2001.


日期:2008年12月28日 - 来自[2002年76卷第3期]栏目

Overexpression of kidney neutral endopeptidase (EC 3.4.24.11) and renal function in experimental cirrhosis

【摘要】  Neutral endopeptidase degrades atrial natriuretic peptide (ANP) and bradykinin and may generate endothelin-1 from big-endothelin. In advanced cirrhosis, sodium retention is accompanied by elevated plasma ANP levels, and infusion of ANP causes hypotension, but in normal humans increasing the concentration of ANP through the inhibition of neutral endopeptidase, localized in renal proximal tubule cells, causes natriuresis without any arterial pressure drop. The purpose of this study was the assessment of kidney neutral endopeptidase expression and responses to candoxatrilat (a specific inhibitor of this enzyme) in rats with CCl 4 -induced cirrhosis. Two groups of control rats ( n = 5) were injected with vehicle or 3 mg/kg candoxatrilat. Three groups of cirrhotic rats with ascites ( n = 10) received vehicle alone or 3 or 10 mg/kg candoxatrilat. In cirrhotic rats, Western blot analysis revealed a 170% increase in renal neutral endopeptidase protein content ( P < 0.03), mainly in the proximal nephron and macula densa, and both candoxatrilat dosages increased plasma ANP levels, urinary volume, and urinary excretion of sodium, ANP, and cGMP compared with vehicle alone (all P < 0.03). Candoxatrilat (10 mg/kg) also reduced tubular solute-free water reabsorption ( P < 0.03) in cirrhotic rats, but renal blood flow, arterial pressure, and plasma renin activity were unaffected. Neutral endopeptidase inhibition has natriuretic and aquaretic actions in cirrhosis without any effect on blood pressure and kidney perfusion due to a significant overexpression of this enzyme in renal cortex.

【关键词】  ascites animal model atrial natriuretic peptide


ENDOGENOUS NATRIURETIC PEPTIDES, atrial natriuretic peptide (ANP), brain-derived natriuretic peptide, C-type natriuretic peptide, and urodilatin, regulate plasma volume and blood pressure ( 43 ). These peptides stimulate natriuresis through the inhibition of the sympathetic nervous system, the renin-angiotensin-aldosterone axis, and AVP secretion and function ( 28 ). ANP clearance occurs mainly in the kidney, lung, brain, and heart and is a result of proteolysis by neutral endopeptidase 24.11 (NEP) and, to a lesser extent, of binding to natriuretic peptide clearance receptors ( 28, 50 ).


Cirrhotic patients with ascites exhibit increased plasma ANP levels and exaggerated peak ANP response during head-out water immersion compared with healthy controls or cirrhotic patients without ascites, due to increased ANP cardiac release ( 14, 35, 38 ). Furthermore, it is of interest that the increase in plasma ANP during head-out water immersion in cirrhosis cannot fully account for the natriuresis of immersion by itself, due to other factors occurring in this setting, i.e., changes in renal-sympathetic nerve traffic and blunting of renin secretion ( 13 ). Because higher ANP levels occur in the setting of sodium retention, this indicates that ascitic patients with cirrhosis exhibit reduced natriuretic responses to this peptide, a finding confirmed by infusion of ANP in cirrhotic patients. Furthermore, ANP infusion determines hypotension in ascitic cirrhosis ( 17 ). The mechanisms of renal hyporesponsiveness to ANP so far identified are activation of the renin-angiotensin-aldosterone axis and sympathoadrenergic tone as a consequence of the arterial hypotensive effect of this hormone ( 49 ), downregulation of natriuretic peptide receptors A and B in the kidney ( 19 ), and increased activity of cGMP phosphodiesterase, the enzyme degrading the second messenger of ANP ( 4 ). The occurrence of renal overexpression of NEP as a possible cause of renal resistance to the effects of ANP has never been investigated in cirrhosis.


NEP is a membrane-bound Zn-metalloendopeptidase of the brush-border membrane of kidney proximal tubule cells, of lung myofibroblasts and epithelial cells, B lymphoid-progenitors, and glial cells ( 2, 25 ). On the surface membrane of these cells, NEP degrades bradykinin ( 11 ), bombesin-like peptide ( 9 ), substance P ( 15 ), ANP ( 23 ), adrenomedullin ( 29 ), endothelin-1 (ET-1) ( 1 ), and angiotensin II ( 15 ). Not only does NEP degrade the above peptides but it produces the vasoconstrictor polypeptide ET-1 from circulating precursors (i.e., big-ET-1 and ET-1 1-31 ) ( 16, 32 ).


NEP is specifically inhibited by candoxatrilat ( 34 ), its prodrug candoxatril ( 3 ), and by thiorphan ( 44 ), sinorphan ( 21 ), and phosphoramidon ( 15 ). In healthy subjects, NEP inhibitors increase the concentrations of ANP and cause natriuresis ( 21, 34 ) without affecting blood pressure ( 21, 34, 39 ). Actually, both candoxatril ( 3 ) and candoxatrilat ( 31 ) have been reported to raise blood pressure in normotensive human subjects.


Because NEP inhibitors potentiate the effects of ANP but do not cause hypotension in other groups of patients (such as those with cardiac failure) ( 34 ), we tested the hypothesis that NEP expression is increased in cirrhosis and that intravenous candoxatrilat would have a natriuretic effect without causing hypotension.


METHODS


Studies were performed in anesthetized adult male Wistar rats with ascitic cirrhosis and anesthetized adult male Wistar control rats. Both groups were fed ad libitum with standard chow and water. Cirrhosis was induced by CCl 4 (Riedel de Haen, Sigma, Seelze, Germany) administered by gavage twice weekly ( 37 ). Cirrhotic rats were studied between 9 and 12 wk after the start of the cirrhosis induction program, when ascites was fully developed. Control rats were studied following a similar period of standardized diet. Experiments in rats were performed in compliance with the procedures outlined in the Italian Ministry of Health guidelines (no. 86/609/EEC) and according to the Principles of Laboratory Animal Care (National Institutes of Health Publication no. 85-23, revised 1985). Candoxatrilat, a specific NEP inhibitor ( 3, 34 ), was provided by Pfizer Central Research (Sandwich, Sussex, UK).


Animal groups. Candoxatrilat was dissolved in 5% glucose solution as diluent to obtain two different solutions to be administered intravenously to the rats in the same volume of fluid (1 ml), producing two different bolus doses, 3 or 10 mg/kg body wt.


The rats were divided into 5 groups: 5 control rats receiving diluent alone, 5 control rats receiving 3 mg/kg body wt candoxatrilat, 10 cirrhotic rats receiving diluent alone, 10 cirrhotic rats receiving 3 mg/kg body wt candoxatrilat, and 10 cirrhotic rats receiving 10 mg/kg body wt candoxatrilat.


Study protocol. The rats were anesthetized with a mixture of Ketavet 100 (Farmaceutici Gellini, Sabaudia, Italy) and Rompum (4:1, vol/vol, Xilazina, Bayer, Leverkusen, Germany) by intraperitoneal injection (0.5 ml mixture/200 g body wt). Blood was sampled ( time 0 ) by cardiac puncture (0.5 ml), and 10% inulin (wt/vol, Laevosan-Gesellschaft, Linz/Donau, Austria) plus 20% PAH (wt/vol, Nephrotest, BAG, Munich, Germany) were administered intravenously into the caudal vein as a priming bolus (0.14 and 0.03 ml/kg, respectively) followed by a continuous infusion of 0.09 ml·kg -1 ·h -1 inulin and 0.025 ml·kg -1 ·h -1 PAH for 150 min to assess glomerular filtration rate (GFR) and renal plasma flow (RPF) at different times by means of their respective steady-state plasma clearances (C In and C PAH ) ( 10, 30 ). After 90 min of inulin plus PAH infusion (i.e., once their steady-state plasma concentrations were reached) ( 10 ), a laparotomy was performed and the urinary bladder was emptied; a clamp was positioned on the urethral orifice. Cardiac blood was then sampled ( time 1 ) to assess basal values of C In and C PAH, and then either diluent alone or candoxatrilat (F 3 or F 10 ) was injected as a single bolus into the right femoral vein. Cardiac blood was then sampled (0.5 ml) at precise intervals (20 min) for 1 h ( times 2-4 ) to measure plasma osmolarity and concentrations of inulin, PAH, sodium, and potassium. At each blood withdrawal, the volume of blood withdrawn was replaced with an equal volume of intravenous saline. Blood samples withdrawn at time 4 (i.e., 60 min after the infusion of diluent or 3 or 10 mg/kg body wt candoxatrilat) were also used to measure plasma concentrations of AVP, ANP, ET-1, and plasma renin activity (PRA). One hour after candoxatrilat or vehicle injection, after collection from the bladder of the urine produced during the 60 min after candoxatrilat or vehicle administration, the rats were killed by exsanguination through the aorta. The urine was used to determine osmolarity and the excretion of sodium, potassium, chloride, ANP, and cGMP. In a further group of five anesthetized cirrhotic rats, mean arterial pressure was evaluated by means of tail sphygmomanometry (Blood Pressure Recorder 8005, W+W Electronic, Milan, Italy) before and 40 min after 10 mg/kg intravenous administration of candoxatrilat in the caudal vein, without performance of a laparotomy.


NEP protein concentration in rat kidneys. For Western blot analysis, membrane fractions were prepared from kidneys removed from five rats in each experimental group (G1-G5); 100-µg slices were homogenized in Tris buffer (20 mM Tris, 2 mM MgCl 2, 0.25 M sucrose, 1 mM PMSF, pH 7.5) and centrifuged at 1,000 g and 4°C for 10 min. The supernatant was centrifuged at 10,000 g for 10 min and at 100,000 g for 45 min. The pellet was dissolved in Tris buffer (50 mM Tris, 2 mM MgCl 2, 80 mM NaCl, 1 mM PMSF, pH 8.0), and the protein content was determined using a modified Bradford assay (Bio-Rad) using BSA as a standard. Protein (15 µg) was loaded on vertical SDS-polyacrylamide gels (4% stacking gel and 10% resolving gel) and transferred to a membrane (Hypobond-PVDF, Amersham Lifescience) by electroblotting in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol). Nonspecific binding was blocked with blocking solution (PBS, 0.05% Tween 20, 5% nonfat dry milk, 5% fetal calf serum), and blots were incubated with a rabbit polyclonal NEP antibody (CD 10, Santa Cruz Biotechnology) and an antibody against the receptor-associated protein (RAP) at a dilution of 1:1,000 in PBS for 1 h at room temperature. After being washed three times for 15 min in PBS, blots were incubated with the secondary antibody (goat IgG, Sigma) for 30 min at room temperature. Three further washing steps (15 min each) in PBS were followed by detection using an ECL detection kit (ECL Western blotting detection, Amersham Pharmacia Biotech). Densitometric quantification was performed using RAP expression as an internal standard: before any comparison was made, the net intensity of NEP bands in each experiment was normalized to the intensity of the corresponding RAP band, used as an internal standard to evaluate the degree of nonspecific protein expression in the homogenate ( 26 ).


NEP immunostaining. Tissue samples were embedded in paraffin, and standard immunohistochemistry procedures were applied using the labeled streptavidin-biotin (LAB-SA) and the AEC-chromogen-producing red staining (Zymed Laboratories) methods. After fixation, endogenous peroxidase activity was quenched with 1% H 2 O 2. Sections were incubated with a primary anti-CD 10-antibody for 16 h (rabbit polyclonal, 2 µg/ml, sc-9149, Santa Cruz Biotechnology). Staining with a nonspecific IgG control antibody (Sigma) served as control.


Plasma and urine analyses. Plasma and urinary concentrations of electrolytes were measured by flame photometry. Inulin and PAH concentrations in plasma were determined colorimetrically ( 40, 46 ). AVP systemic concentrations were measured in EDTA plasma by RIA (Vasopressin Direct RIA, Buhlmann Laboratories, Postfach, Switzerland). Urine and plasma ANP was measured using a specific RIA (ANP Shionoria, Cis Bio International, Gif-sur-Yvette Cedex, France). PRA was determined using RIA for angiotensin I after an incubation period of 2 h (Renin Maia Kit, Biodata, Rome, Italy). ET-1 plasma concentrations were measured using a commercially available kit (Endothelin-1 Radioimmunoassay, Peninsula Laboratories, King of Prussia, PA). Urinary concentrations of cGMP were determined using a commercial RIA kit (Immunotech, Marseille, France).


Calculations. Sodium clearance (C Na ) and potassium clearance (C K ) were calculated through the formula


where U x is the urinary concentration of x, P x is the plasma concentration of x, and V is the urinary output (ml/min). C In and C PAH were calculated through the steady-state plasma clearance formula as


where ssP x is the steady-state plasma concentration of x. C In and C PAH were taken as measures of GFR and RPF ( 30, 31 ). Filtered sodium load (Fl Na ) was derived, following Boer et al. ( 6 ), as


Filtration fraction (FF) was calculated from the formula


Fractional sodium excretion (FE Na ) and fractional potassium excretion (FE K ) were calculated, respectively, from the ratios of C Na and C K to C In x 100.


Tubular solute-free water reabsorption (TFWR) was calculated, following Rose and Post ( 41 ), through the formula


where V is urinary output (ml/min), and C osm is the osmolar clearance, which was computed via the usual formula


where U osm and P osm are urine and plasma osmolarities, respectively.


All renal function parameters measured after diluent or candoxatrilat administration were derived by computing the mean of three determinations of osmolarity, inulin, PAH, sodium, and potassium in plasma during the 60-min urine collection period (blood sampling times 2-4 ).


Mean arterial pressure (MAP) was calculated from the formula


Morphological liver studies. Livers were removed from 20 rats submitted to CCl 4 intoxication, and hepatic tissue samples for light microscopy were placed in buffered 4% formaldehyde solution (pH 7.4). The sections were stained with hematoxylin and eosin to assess fibrosis. Silver-impregnated liver sections were used to observe portal-central or central-central bridging fibrosis.


Statistical analysis. The main comparisons were between renal or hormonal parameters measured after administration of candoxatrilat or after diluent alone. Results are expressed as means ± SD. All comparisons between groups of rats were made by a nonparametric statistical method, the Wilcoxon rank sum test. Correlation coefficients were derived using Spearman's rank correlation. Significance is accepted at the 5% probability level.


RESULTS


Liver morphological studies. Micronodular cirrhosis with hepatocellular necrosis and microvacuolar steatosis was found in all 20 livers removed from ascitic rats (data not shown).


Renal NEP expression. Neutral endopeptidase appeared significantly overexpressed in the membrane fraction of renal tissue homogenate from cirrhotic animals, without differences in expression between candoxatrilat-treated and untreated animals ( Figs. 1 and 2 ). Immunohistochemical determination of NEP in renal tissue slices showed more intense positive NEP staining in the proximal convoluted tubules, the macula densa, Bowman's capsule, and the mesangium in kidneys from rats with liver cirrhosis compared with controls ( Fig. 3 ).


Fig. 1. Western blot of representative experiments showing neutral endopeptidase (NEP) levels in the membrane fractions of kidneys of control rats (C), cirrhotic rats given vehicle (Cir), and cirrhotic rats given candoxatrilat 10 mg/kg body wt (F10). Receptor-associated protein (RAP) is an internal standard used to evaluate the degree of nonspecific protein expression in this homogenate.


Fig. 2. Relative densities of NEP bands. Values are means ± SD of 5 rats/group.


Fig. 3. Immunohistochemical determination of NEP in kidney cortex from a control ( A ) and a cirrhotic rat ( B ). A more intense positive reaction was observed in proximal convoluted tubules, macula densa (arrow), Bowman's capsule, and mesangium in kidneys from rats with liver cirrhosis ( x 400, hematoxylin counterstained).


Hormonal status and mean arterial pressure. Infusion of candoxatrilat caused a significant increase in plasma ANP concentrations in both control and cirrhotic rats but had no effect on plasma renin activity, ET-1, or AVP levels ( Table 1 ). Both candoxatrilat dosages significantly raised urinary excretion of ANP ( Fig. 4 ) and cGMP in cirrhotic animals. Cirrhotic rats infused with candoxatrilat had a significantly lower urinary cGMP/sodium ratio, suggesting that potentiation of natriuretic peptide function is not the sole mechanism that increases natriuresis after NEP inhibition. Candoxatrilat had no effect on blood pressure [89 ± 27 vs. 86 ± 37 mmHg ( P = 0.37)] in cirrhotic rats.


Table 1. Hormonal plasma and urine determinations in different groups of rats


Fig. 4. ANP urinary excretion values. G1, control rats receiving diluent alone; G2, control rats receiving 3 mg/kg body wt candoxatrilat; G3, cirrhotic rats receiving diluent alone; G4, cirrhotic rats receiving 3 mg/kg body wt candoxatrilat; G5, cirrhotic rats receiving 10 mg/kg body wt candoxatrilat.


Renal function. In cirrhotic rats, both 3 and 10 mg/kg candoxatrilat caused marked diuresis and increased absolute ( Fig. 5 ) and fractional excretion of sodium; a significant reduction of tubular solute-free water reabsorption and urinary osmolarity was observed after 10 mg/kg candoxatrilat ( Table 2 ). Because FE Na was also moderately decreased in the control group receiving vehicle alone, a certain degree of reduction of the circulating fluid volume due to the experimental procedures cannot be ruled out; the increased urine osmolarity and tubular free water reabsorption observed in the cirrhotic group confirm the occurrence of free water retention in those rats. Candoxatrilat had no effect on RPF, GFR, or FF.


Fig. 5. Sodium urinary excretion values. Abbreviations are defined as in Fig. 4.


Table 2. Renal function data after administration of candoxatrilat or diluent alone


Correlations. In both controls and cirrhotic rats, there were significant correlations between FF and diuresis (respectively, r = 0.61, P < 0.05 and r = 0.43, P < 0.03) and between urinary excretions of cGMP and natriuresis (respectively, r = 0.68, P < 0.05 and r = 0.70, P < 0.001) ( Fig. 6 ). However, there was a significant correlation between ANP and cGMP urinary excretions only in the control group receiving diluent alone ( r = 0.79, P < 0.05) but in none of the other groups of rats. We found a significant correlation between urinary excretion of sodium and ANP only in control or cirrhotic rats given diluent alone (respectively, r = 0.84, P < 0.05 and r = 0.79, P < 0.001) but not in candoxatrilat-treated groups.


Fig. 6. Cirrhotic rats (30 animals). There was a significant direct correlation between urinary excretion of cGMP and that of sodium.


DISCUSSION


In advanced cirrhosis, splanchnic vasodilatation activates the renin-angiotensin-aldosterone axis, sympathetic nervous system, and nonosmotic hypersecretion of AVP, with ensuing renal sodium and water retention and ascites development ( 8, 22 ). This sodium and water retention is treated with diuretic drugs that determine a further stimulation of renin secretion and may even lead to prerenal azotemia. Therefore, a drug able to enhance exclusively the renal vasodilator, natriuretic, and AVP-antagonistic actions of ANP but be devoid of the systemic hypotensive effects of this hormone would be of outstanding importance in treating cirrhotic ascites.


The effects of NEP inhibitors in cirrhosis have been evaluated in two previous studies. Dussaule et al. ( 12 ) administered single oral doses of sinorphan to 16 patients with ascitic cirrhosis and observed increases in plasma ANP, a natriuretic response, a significant reduction of plasma renin activity, and no effects on arterial pressure. Park et al. ( 36 ) observed that thiorphan caused natriuresis but did not alter cardiac output and systemic vascular resistance in cirrhotic rats. Neither study evaluated the effects of NEP inhibition on renal perfusion and free water metabolism, AVP plasma levels, and ANP urinary excretion. Finally, the kidney biomolecular expression of NEP in cirrhosis has never been investigated.


We observed that NEP inhibition with candoxatrilat significantly increased ANP urinary excretion in rats with ascitic cirrhosis ( Fig. 4 ) while causing a smaller increase in plasma ANP ( Table 1 ), leading to increased cGMP and sodium urinary excretion rates ( Fig. 5 ) and decreased tubular solute-free water reabsorption. No effects on mean arterial pressure, PRA, and AVP secretion were found ( Tables 1 and 2 ). These results confirm that if delivery of ANP to the renal tubules is increased, ANP effects may be targeted to the required site.


The increase in FE Na produced by candoxatrilat, without any change in GFR, suggests exclusive tubular actions of this drug. It has been proposed that NEP inhibitors might decrease proximal tubular reabsorption of sodium and water ( 7, 15 ), because, in isolated microperfused tubule models, ANP decreases angiotensin II-stimulated proximal tubular sodium reabsorption ( 18, 24 ). The aquaretic action of 10 mg/kg candoxatrilat ( Table 2 ) strengthens this assumption, as any increase in distal tubular delivery of fluid enhances the capacity of the cortical portion of the ascending limb of Henle to generate free water ( 42 ).


Further tubular diuretic mechanisms of candoxatrilat probably exist: 1 ) NEP inhibitors may reduce intrarenal levels of sodium-retaining factors ( 45 ) because enhancement of ANP tubular content by NEP inhibition limits renin secretion by targeting the macula densa ( 12, 23 ); 2 ) urinary ANP and natriuresis were correlated only when control or cirrhotic rats were given vehicle, but not in the candoxatrilat-treated groups; 3 ) compared with cirrhotic rats receiving vehicle, the candoxatrilat-treated cirrhotic groups had higher urinary sodium excretions for any given value of cGMP urinary excretion ( Table 1 ); and 4 ) when inhibition of NEP is accompanied by administration of bradykinin or adrenomedullin antagonists, blunted natriuretic effects are found ( 11, 29 ). Indeed, in the model of dogs with ascites due to chronic constriction of the supradiaphragmatic vena cava (chronic caval dogs), it was postulated that, during NEP inhibition, increased delivery of both kinins and ANP distally to the cortical collecting duct allowed a natriuresis in the animals resistant to the sole infusion of exogenous ANP ( 27 ). Last, NEP inhibition might also account for decreased degradation of kaliuretic peptides derived from ANP prohormone ( 48 ), because candoxatrilat determined a strong tendency toward increased absolute potassium excretion and FE K ( Table 2 ).


In the kidneys of cirrhotic rats, we found overexpression of NEP ( Figs. 1 and 2 ), mainly in the proximal convoluted tubules, macula densa, Bowman's capsule, and mesangium ( Fig. 3 ). Consequently, ANP urinary excretion in cirrhotic rats was significantly lower than in either group of healthy animals ( Table 1 ) despite similar GFR values ( Table 2 ). This finding, previously described in chronic caval dogs with ascites ( 27 ), confirms enhanced tubular degradation of ANP in cirrhosis. Accordingly, the lower candoxatrilat dose we used caused a significant increase in absolute sodium excretion and FE Na only in rats with cirrhosis ( Table 2 and Fig. 5 ). NEP overexpression has been found to contribute to ANP resistance also in rats with congestive heart failure ( 26 ).


The cause of NEP overexpression in cirrhotic rat kidneys is unknown; however, several cytokines whose systemic levels are increased in liver cirrhosis ( 47 ), interleukin-1, tumor necrosis factor-, transforming growth factor-, and interleukin-6, enhance the in vitro expression of NEP on the surface of fibroblasts and bronchial and kidney tubular epithelial cells ( 3, 9 ).


In agreement with the findings of Park et al. ( 36 ), in our study NEP inhibition did not cause significant changes in ET-1 plasma levels in cirrhotic rats. Previous studies have reported widespread results: Newaz et al. ( 33 ) found a reduction in plasma ET-1 after candoxatril administration in arterial hypertensive rats. Asaad et al. ( 5 ) reported that NEP did not contribute to the in vivo clearance of ET-1 in rats, whereas in a study in rabbits Grantham et al. ( 20 ) demonstrated a reduction in aortic tissue concentration of ET-1, despite plasma concentrations of ET-1 being increased, after NEP inhibition. These variable results are explained by the curious behavior of NEP, an enzyme that, depending on location and available substrates, can inactivate ET-1 or produce this hormone via hydrolysis of big-ET-1 and ET-1 (1-31) ( 32 ).


Our study has identified a new target for future therapeutic regimens aimed at treating patients with liver cirrhosis and sodium and water retention. Furthermore, improved understanding of the subtle biohumoral modifications occurring in the kidney after inhibition of neutral endopeptidase may provide a valid occasion for knowledge of this clinical syndrome to advance further.


GRANTS


This study was supported by grants from the Italian Ministry of Universities and of Scientific Research (60%), 2001.


ACKNOWLEDGMENTS


We thank Dr. Kevin Moore (Centre for Hepatology, Royal Free and University College Medical School, University College London) for the preliminary revision of this paper.


This study was presented orally at the following meetings: 2003 Digestive Disease Week, Orlando, FL, May 2003; 39th meeting of the European Association for the Study of the Liver, Berlin, Germany, April 2004; and the 55th meeting of the American Association for the Study of Liver Diseases, Boston, MA, October 2004.

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作者单位:1 Gastroenterology Unit, Gradenigo Hospital, 2 Department of Experimental Medicine and Oncology, 3 Department of Clinical and Biological Sciences, 4 Department of Gastroenterology, and 5 Institute of General Pathology, Molinette Hospital, University of Turin, Turin, Italy

日期:2008年7月4日 - 来自[2006年第289卷第6期]栏目

New Clues for Liver Cirrhosis Treatment

Dec. 27, 2007 -- Working with mice, researchers have found a molecule that prevents -- and even reverses -- formation of scar tissue in damaged livers.

The finding promises new treatments for cirrhosis and other scarring diseases of the liver, and perhaps for other scarring-related conditions such as pulmonary fibrosis, scleroderma, and burns.

Livers damaged by disease, toxins, or injury tend to develop excessive scar tissue -- a condition called liver fibrosis. This process lies at the heart of cirrhosis, in which bands of scar tissue overgrow the liver. There's currently no sure way to prevent or reverse this process once it's begun.

But excessive scarring happens only when a protein called RSK is activated in liver cells, find Martina Buck, PhD, and colleagues at the University of San Diego and the San Diego VA Healthcare System.

Mice genetically engineered to produce an RSK-blocking peptide did not develop liver fibrosis when poisoned with a liver toxin. And when the peptide was injected into normal mice, it protected them against the liver toxin.

"All control mice had severe liver fibrosis, while all mice that received the RSK inhibitory peptide had minimal or no liver fibrosis," Buck says in a news release.

Scar tissue is made up of a natural material called collagen. Liver cells called hepatic stellate cells (HSCs) don't make much collagen unless activated by the stress of injury or disease. Once activated, however, these cells make way too much collagen. The result: scar tissue.

The RSK inhibitory peptide causes these activated HSCs to self-destruct, while normal liver cells continue to heal the liver.

"Remarkably, the death of HSCs may also allow recovery from injury and reversal of liver fibrosis," Buck says.

Human HSCs work much the same way as mouse HSCs, so the findings should apply to human disease, the researchers suggest.

Buck and colleagues are hopeful that the RSK inhibitory peptide will be the model for a future human drug.

"We speculate that these findings may facilitate the development of small molecules potentially useful in the prevention and treatment of liver fibrosis," Buck and colleagues conclude. "Blocking the progression of liver fibrosis would decrease development of primary liver cancer in these patients since the majority of [liver cancers] arise in cirrhotic livers."

Buck and colleagues report their findings in the Dec. 26 issue of the online journal PloS One.

日期:2007年12月31日 - 来自[Health News]栏目

Coffee Might Curb Alcoholic Cirrhosis

June 12, 2006 -- Coffee may contain an ingredient that protects the liver against alcoholic cirrhosiscirrhosis, a new study shows.

The study, published in the Archives of Internal Medicine, shows that among more than 125,000 people studied for up to 22 years, coffee drinkers were less likely to be diagnosed with alcoholic cirrhosis.

"These data support the hypothesis that there is an ingredient in coffee that protects against cirrhosis, especially alcoholic cirrhosis," the researchers write.

But they aren't recommending that anyone rely on coffee to prevent alcoholic cirrhosis. Not drinking heavily is a better strategy for liver health, the researchers note.

They included Arthur Klatzky, MD. He works in the research division of Kaiser Permanente Medical Care Program of Oakland, Calif.

Coffee and Alcohol

Participants signed up for the study between 1978 and 1985. They were followed until the end of 2001.

Upon joining the study, participants got a checkup and completed surveys about their use of alcohol, coffee, tea, and cigarettes. None had already been diagnosed with liver problems.

Most participants noted drinking light or moderate amounts of alcohol (up to two daily drinks). Only 8% admitted drinking three or more alcoholic drinks per day.

Typical coffee consumption was one to three daily cups, noted by 42% of the group. Another 16% reported drinking four or more daily cups of coffee.

Alcoholic Cirrhosis Rarer

Over the years, 330 participants were diagnosed with cirrhosis; 199 of those cases were alcoholic cirrhosis. The remaining cases of cirrhosis were nonalcohol related.

For every daily cup of coffee that participants reported drinking, they were 22% less likely to have been diagnosed with alcoholic cirrhosis during the study, Klatzky's team reports.

The odds of developing nonalcoholic cirrhosis weren't linked to coffee consumption.

Coffee drinkers were also less likely to have high blood levels of liver enzymes. That pattern was strongest in people with the highest reported alcohol consumption.

Participants only took the survey once -- when the study started. So the data don't include changes in coffee or alcohol consumption.

People don't always report their drinking habits accurately, note Klatzky and colleagues, adding that participants' alcohol use is "relatively stable."

Was It the Caffeine?

Klatzky's team didn't identify what ingredient in coffee might help protect the liver from alcoholic cirrhosis.

Caffeine might not get the credit. Tea contains caffeine, but tea consumption didn't appear to lower participants' odds of being diagnosed with any form of cirrhosis.

Past studies on caffeine and alcoholic cirrhosis haven't reached any firm conclusions, note Klatzky and colleagues. "In our opinion this issue is quite unresolved," they write, adding that coffee drinkers were more common than tea drinkers in their study.

As an observational study, the study doesn't prove that drinking coffee cuts the chance of developing alcoholic cirrhoses, the researchers caution. They also point out that if coffee protects the liver, the effects of adding cream, milk, sugar, or other substances to coffee aren't yet known.


SOURCES: Klatsky, A. Archives of Internal Medicine, June 12, 2006; vol 166: pp 1190-1195. News release, JAMA/Archives. News release, Kaiser Permanente.

日期:2006年7月4日 - 来自[General Health]栏目
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