The influence of urinary flow rate on mercury excretion in children

ProjectThere is limited literature concerning the effect of urinary flow rate on mercury excretion at low-level exposure. The aim of the present study is to examine the influence of urinary flow rate on mercury excretion in children. Also of interest is the influence of flow rate on creatinine excretion and creatinine-corrected mercury, which arisearises with spot urine samples.ProcedureA substudy of the New England Children's Amalgam Trial collected pairs of urine samples from children aged 10–16 years: a timed overnight collection and a spot daytime sample collected the following day. These samples were analyzed for mercury and creatinine concentration. Regression analysis was used to model the effect of urinary flow rate in the timed overnight samples. A paired t-test compared concentrations and creatinine-corrected mercury between overnight and daytime samples.ResultsCreatinine excretion rate (mg/h) increased significantly with urinary flow rate (mL/h), whereas creatinine concentration (g/L) decreased with flow rate. We found a non-significant increase in mercury excretion rate (ng/h) with flow rate, and mercury concentration decreased with flow rate. Mercury and creatinine concentrations were significantly higher in the overnight compared to daytime samples. For creatinine-corrected mercury, no significant impact of urinary flow rate was found.ConclusionsAlthough the creatinine excretion rate, and probably the mercury excretion rate, increased with urinary flow rate, the mercury/creatinine ratio seemed relatively unaffected by urinary flow rate.     

大鼠尿中酶类参考值检测

目的调查正常大鼠尿液样本中酶类参考值范围。方法采集95只正常SD大鼠的4 h尿液,用生化仪检测尿液中的N-乙酰基-β-D氨基葡萄糖酶（NAG）、碱性磷酸酶（ALP）、谷氨酰氨基转移酶（γ-GT）和乳酸脱氢酶（LDH）的值,参考范围以95%百分位上界表示。结果参考值范围为,雌性：NAG〈5.3 U/L,ALP〈110.8 U/L,γ-GT〈753.0 U/L,LDH〈32.6 U/L;雄性：NAG〈27.3 U/L,ALP〈329.0 U/L,γ-GT〈769.8 U/L,LDH〈40.0 U/L。以/g Cre为单位的参考值范围,雌性：NAG〈28.9 U/g,ALP〈723.5 U/g,γ-GT〈1763.2 U/g,LDH〈113.1 U/g;雄性：NAG〈84.3 U/g,ALP〈680.2 U/g,γ-GT〈2522.3 U/g,LDH〈121.8 U/g。结论尿中酶类参考值范围为药物肾毒性评价提供了背景数据。     

Quantitation of plasma thiamine,related metabolites and plasma protein oxidative damage markers in children with autism spectrum disorder and healthy controls

Abstract

Aims/hypothesis: To assess thiamine and related metabolite status by analysis of plasma and urine in autistic children and healthy controls, correlations to clinical characteristics and link to plasma protein markers of oxidative damage.

Methods: 27 children with autism (21 males and 6 females) and 21 (15 males and 6 females) age-matched healthy control children were recruited. The concentration of thiamine and related phosphorylated metabolites in plasma and urine and plasma protein content of dityrosine, N-formylkynurenine and 3-nitrotyrosine was determined.

Results: Plasma thiamine and thiamine monophosphate concentrations were similar in both study groups (median [lower–upper quartile]): autistic children – 6.60?nM (4.48–8.91) and 7.00?nM (5.51–8.55), and healthy controls – 6.82?nM (4.47–7.02) and 6.82?nM (5.84–8.91), respectively. Thiamine pyrophosphate (TPP) was decreased 24% in autistic children compared to healthy controls: 6.82?nM (5.81–8.52) versus 9.00?nM (8.41–10.71), p?<?.01. Urinary excretion of thiamine and fractional renal clearance of thiamine did not change between the groups. No correlation was observed between clinical markers and the plasma and urine thiamine concentration. Plasma protein dityrosine content was increased 88% in ASD. Other oxidative markers were unchanged.

Conclusions/interpretation: Autistic children had normal plasma and urinary thiamine levels whereas plasma TPP concentration was decreased. The latter may be linked to abnormal tissue handling and/or absorption from gut microbiota of TPP which warrants further investigation. Increased plasma protein dityrosine may reflect increased dual oxidase activity in response to change in mucosal immunity and host–microbe homeostasis.     

γ-Tocopherol biokinetics and transformation in humans

Background: The uptake and biotransformation of γ-tocopherol (γ-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of γ-T biology in healthy volunteers and their response to γ-T supplementation.

Methods: A single bolus of 100 mg of deuterium labeled γ-T acetate (d²-γ-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d² and d⁰-γ-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-γ-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of γ-T supplementation on α-T concentrations in plasma and α-T and γ-T metabolite formation were also assessed by HPLC or GC-MS analysis.

Results: At baseline, mean plasma α-T concentration was approximately 15 times higher than γ-T (28.3 vs. 1.9 µmol/l). In contrast, plasma γ-CEHC concentration (0.191 µmol/l) was 12 fold greater than α-CEHC (0.016 µmol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 µmol, respectively) suggesting that the clearance of α-CEHC from plasma was more than 40 times that of γ-CEHC. After d²-γ-TAC administration, the d² forms of γ-T and γ-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d⁰ species were hardly affected. Mean total concentrations of γ-T and γ-CEHC in plasma and urine peaked, respectively, between 0–9, 6–12 and 9–24 h post-supplementation with increases over baseline levels of 6–14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d²-γ-T equivalents was approximately 7 mg. Baseline levels of γ-T correlated positively with the post-supplementation rise of (d⁰ + d²) – γ – T and γ-CEHC levels in plasma, but correlated negatively with urinary levels of (d⁰ + d²)-γ-CEHC. Supplementation with 100 mg γ-TAC had minimal influence on plasma concentrations of α-T and α-T-related metabolite formation and excretion.

Conclusions: Ingestion of 100mg of γ-TAC transiently increases plasma concentrations of γ-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of γ-T nor the concentration and metabolism of α-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios α-T to γ-T and γ-CEHC to α-CEHC.     

Role of hippurate in acidosis induced adaptation in renal gamma-glutamyltransferase

Metabolic acidosis results in an adaptation in renal γ-glutamyltransferase (γ-GT) and a doubling of hippurate excretion. The greater rate of γ-glutamohydroxamate, γ-GHA, formation from L-glutamine, but not from glutathione, by acidotic kidney homogenates suggest an increased γ-glutamyl-enzyme complex formation and a preference for glutamine as the γ-glutamyl donor in acidosis. Hippurate added $\text{in}$$\text{vitro}$ to cortical homogenates or microsomes mimics the affect of acidosis upon γ-GHA formation from glutamine. Acid extracts of urine stimulated ammonia formation from glutamine using cortical microsomes in agreement with the measured hippurate levels. Administering an exogenous hippurate load to fasting nonacidotic rats doubled ammonia excretion and the rate of γ-GHA formation by cortical homogenates. These results are consistent with the acidosis induced adaptation in renal γ-GT governed by hippurate.     

Increased urinary zinc excretion in cancer patients is linked to immune activation and renal tubular cell dysfunction

Urinary zinc excretion is known to be increased in cancer patients, but the pathogenesis of this phenomenon remains uncertain. Both skeletal muscle catabolism and renal tubular cell dysfunction have been proposed to explain this observation. We have investigated urinary zinc and N-acetyl--d-glucosaminidase (NAG), an indicator of renal tubular cell dysfunction, as well as serum neopterin, an index of systemic immune activation, in 22 patients with cancer and seven controls. Both serum neopterin and urinary zinc were significantly elevated in cancer patients (15.8 ± 12.7 versus 7.3 ± 2.3 nmol l^–1 and 1.77 ± 0.80 versus 1.21 ± 0.41 mmol mol^–1 creatinine, P < 0 and P < 0.05, respectively), while NAG was similar in cancer patients and the controls (13.58 ± 13.80 versus 13.68 ± 12.19 kat mol^–1 creatinine). A significant correlation was observed between serum neopterin and urine zinc (rs = 0.5119, P < 0.02), serum neopterin and urine NAG (rs = 0.6761, P < 0.002), and urinary zinc and NAG (rs = 0.6348, P < 0.002). In conclusion, the present data indicate a link between urinary zinc excretion and immune activation as well as renal tubular cell dysfunction. In addition, renal tubular cell dysfunction appears to be linked to immune activation.     

Age related changes of N-acetyl-beta-D-glucosaminidase and L-alanine aminopeptidase in mouse kidney, urine and plasma

Age and sex dependent differences of N-acetyl-beta-D-glucosaminidase (NAG) and L-alanine aminopeptidase (AAP) activities in kidney, urine and plasma of male and female mice were studied. The sex difference in NAG activity appeared between 27 and 38 days of age with the manifestation of significant differences in body weight and kidney growth. NAG activity in male kidneys was 3-fold that in females and its urinary level in mature males was over 10-fold higher. Androgenic regulation was found not only in the NAG contents in the kidneys and in the urinary excretion but also in the plasma NAG level, which showed higher in females. On the other hand, AAP activity in kidney, urine and plasma did not show much sex differences. Age related changes in AAP activity were not found except in the kidney and marked androgenic regulation was also not found in AAP. These results indicate that NAG and AAP, which are both urinary enzymes used as indicators of renal lesions, may be regulated differently.     

Urinary 8-hydroxy-2′-deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection

Abstract

Background: There are no good biomarkers to predict renal parenchymal involvement in children with urinary tract infection (UTI).

Methods: Children (N?=?73) younger than 5 years with UTI were enrolled. Urinary levels of 8-hydroxy-2′-deoxyguanosine (8-oxodG) and total antioxidant capacity (TAC) were checked as markers of oxidative stress and antioxidant capacity, respectively. Tc99m-dimercaptosuccinic acid (DMSA) renal scintigraphy was used to find evidence of renal involvement.

Results: Patients with positive DMSA findings had higher levels of urinary 8-oxodG (p?=?0.003) and higher urinary TAC (p?=?0.001) than patients with normal DMSA findings.

Conclusions: High level of urinary 8-oxodG may be a risk factor of severe renal damage.     

Fate of mercury in a terrestrial biological lab process using Polypogon monspeliensis and Cyperus odoratus

Abstract

Mercury has been extracted in Queretaro, Mexico since the 1960s. The mining wastes were open-air disposal and these mercury wastes have polluted the zone. The aim of this research was to evaluate mercury's fate in lab scale terrestrial reactors considering the following mercury species: soluble, interchangeable, strongly bound, organic, and residual ones. Soils were sampled in two former mines of Pinal de Amoles, Queretaro, Mexico (N 20° 58′ to 21° 21′ and West 99° 26′ to 99° 43′) with initial mercury concentrations were 424?±?29 and 433?±?12?mg?kg^?1 for “La Lorena” and “San Jose” former mines, respectively. Two vegetal species Polypogon monspeliensis and Cyperus odoratus were used and 20 reactors were constructed for the lab process. Total mercury was removed to 49–79% from both soils. Mercury elemental, exchangeable, and organic species had the most removal or exchange in the process. Metal uptake, by the plants, was of 5–6% for P. monspeliensis and 5–15% for C. odoratus. Also, mercury fate was estimated to the atmosphere to be 3.3–4.5?mg m^?2 h^?1 for both soils.     

Association between urinary indicators of renal dysfunction and metal concentrations in workers chronically co-exposed to cadmium,zinc and lead

The study was carried out in 31 workers co-exposed to cadmium, lead and zinc fumes and dusts in a zinc ore refinery. Urinary cadmium, lead, zinc, β₂-M levels and NAG activities were determined to evaluate the possible dose-effect relationship between these parameters. A correlation was found between urinary cadmium, lead and zinc concentrations, and urinary β₂-M levels and NAG activities of the exposed group. A statistically significant increase was also observed for urinary NAG activity in exposed workers who had urinary cadmium concentrations > 2 μg g^?1 creatinine. However, in the same exposed group, the increment of β₂-M was not statistically significant. In conclusion, the present study thus confirms the earlier observations and may suggest the notion that the urinary NAG seems to be a more sensitive indicator than urinary β₂-M level in early stages of renal injury of moderately cadmium co-exposure with lead and zinc even at urinary cadmium concentration as low as 2 μg g^?1 creatinine. When the earlier studies on the irreversibility of cadmium-induced tubular dysfunction and the present results were taken into consideration, the present health-based biological limit proposed by the WHO (5 μg g^?1 creatinine) seems to be high for the occupational exposure to cadmium.     

Mortality prediction by acute kidney injury biomarkers in comparison with serum creatinine

Abstract

Objectives: To investigate the performance of acute kidney injury (AKI) biomarkers for mortality prediction.

Materials and methods: Cutoff values of urinary L-type fatty acid-binding protein (L-FABP) and N-acetyl-β-d-glucosaminidase (NAG) for AKI diagnosis in ICU were determined in the derivation cohort. The performance of these AKI biomarkers for mortality prediction was evaluated in the validation cohort with stratification of serum-creatinine based AKI diagnosis.

Results: Mortality in the AKI patients diagnosed by serum creatinine was increased remarkably when urinary L-FABP and NAG were positive.

Conclusions: These AKI biomarkers can specifically detect high-risk patients among creatinine-base diagnosed AKI.     

Urinary enzyme concentrations in the owl monkey (Aotus nancymae)

The activity of three urinary enzymes, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and N-acetyl-β-D-glucosaminidase (NAG), was evaluated in 71 adult owl monkeys. Fifty-six animals had normal renal function, while 15 had evidence of renal dysfunction. Urinary enzyme: urinary creatinine ratios (U_E:U_Cr) were also determined. The activity for NAG was similar to that of other species, while ALP and AST were higher. Regression analyses revealed that urinary enzymes and U_E:U_Cr were significantly correlated (P ≤ 0.0001) with indices of renal damage and could identify active renal disease.     

Urinary oxidative stress markers in children with autism

Abstract

Oxidative stress caused by increased production of free radicals and impaired functions of antioxidants remains as the major factor associated with the pathophysiology of many neuropsychiatric diseases.

Objective

The objective of the present study was to analyze the oxidative stress markers in urine sample since the collection of blood from these children is highly meticulous and also to evaluate whether these urinary markers can be correlated with the severity of autism.

Methods

The subjects of the study were 45 autistic children with different grades of severity (low functioning autism (LFA), medium functioning autism (MFA), and high functioning autism (HFA) according to Childhood Autism Rating Scale (CARS), n = 15 children in each group and 50 healthy children (age and sex matched). The boys and girls ratio involved in this study was 4:1, and they were of age 4–12 years. We determined the urinary levels of oxidative stress markers like thiobarbituric acid-reacting substances, lipid hydroperoxides, 4-hydroxy nonenal, protein carbonyls, sulfhydryl groups, total antioxidant capacity, total peroxide content, oxidative stress index, and also UA/Cr ratio in autistic children.

Results

The study observed a significant elevation in the level of oxidative stress markers in autistic children when compared with normal children. The level of antioxidants excreted in urine was found to be significantly low in autistic children. These findings when correlated with the degrees of severity, oxidative stress markers showed positive correlation with increasing order of severity (LFA > MFA > HFA), whereas antioxidants showed negative correlation.

Discussion

The study reveals that the urinary levels of oxidative stress markers can be considered as the measure of oxidative stress index in autistic children. The significant correlation between the severity of autism with urinary lipid peroxidation products also support the use of oxidative stress markers and antioxidants as biomarkers of autism.     

Effect of polyaspartic acid on CdCl2-induced nephrotoxicity in the rat

We produced an animal model of CdCl₂ nephrotoxicity in rats, and treated them with polyaspartic acid (PAA) to prevent renal damage. Male Sprague-Dawley (SD) rats (190–200 g) were used to induce proximal renal tubular damage by daily injection of CdCl₂ 3.0 mg/1,000 g body wt for 2 wk. CdCl₂-exposed SD rats exhibited significant increases in urine volume, urinary excretion ofN-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and fractional excretion of sodium (FENa) and a decrease in the percentage of tubular reabsorption of phosphate (%TRP). Of these indicators of proximal tubular function, AAP and %TRP are more sensitive than NAG or FENa. No glycosuria or aminoaciduria, however, were observed. PAA markedly improved these indicators of proximal tubular function. Daily urinary protein excretion and creatinine clearance, on the other hand, did not change after administration of PAA. Cd concentrations in the cortex were 3 times higher than in the medulla, however, there were no differences between Cd-treated rats and PAA-treated rats. Our animal model is an excellent one for determining the effect of cadmium on renal proximal tubule damage. PAA appears to be useful in the treatment of CdCl₂ nephrotoxicity.     

Reliability of urine lactate as a novel biomarker of lactate production capacity in maximal swimming

Context: Postexercise urine lactate may be a novel biomarker of lactate production capacity during exercise.

Objective: To evaluate the reliability and utility of the urine lactate concentration after maximal swimming trials between different training protocols (6?×?50?m and 3?×?100?m) and training states (active and nonactive swimmers).

Materials and methods: Lactate and creatinine were determined by spectrophotometry in blood and urine.

Results: Blood and urine lactate concentrations were correlated in-between training protocols and in participants of different training states. The reliability of the urine lactate concentration was moderate for one of the training protocols and good or moderate for the two training states. Additionally, it was lower than that of the blood lactate concentration, and did not improve after normalizing to the urine creatinine concentration.

Discussion and conclusion: Although promising as a biomarker of lactate production capacity, urine lactate requires further research to improve its reliability.     

A novel antioxidant agent caffeic acid phenethyl ester prevents long-term mobile phone exposure-induced renal impairment in rat

Caffeic acid phenethyl ester (CAPE), a flavonoid like compound, is one of the major components of honeybee propolis. It has been used in folk medicine for many years in Middle East countries. It was found to be a potent free radical scavenger and antioxidant recently. The aim of this study was to examine long-term applied 900 MHz emitting mobile phone-induced oxidative stress that promotes production of reactive oxygen species (ROS) and, was to investigate the role of CAPE on kidney tissue against the possible electromagnetic radiation (EMR)-induced renal impairment in rats. In particular, the ROS such as superoxide and nitric oxide (NO) may contribute to the pathophysiology of EMR-induced renal impairment. Malondialdehyde (MDA, an index of lipid peroxidation) levels, urinary N-acetyl-β-d-glucosaminidase (NAG, a marker of renal tubular injury) and nitric oxide (NO, an oxidant product) levels were used as markers of oxidative stress-induced renal impairment and the success of CAPE treatment. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in renal tissue were determined to evaluate the changes of antioxidant status. The rats used in the study were randomly grouped (10 each) as follows: i) Control group (without stress and EMR), ii) Sham-operated rats stayed without exposure to EMR (exposure device off), iii) Rats exposed to 900 MHz EMR (EMR group), and iv) A 900 MHz EMR exposed + CAPE treated group (EMR + CAPE group). In the EMR exposed group, while tissue MDA, NO levels and urinary NAG levels increased (p < 0.0001), the activities of SOD, CAT, and GSH-Px in renal tissue were reduced (p < 0.001). CAPE treatment reversed these effects as well (p < 0.0001, p < 0.001 respectively). In conclusion, the increase in NO and MDA levels of renal tissue, and in urinary NAG with the decrease in renal SOD, CAT, GSH-Px activities demonstrate the role of oxidative mechanisms in 900 MHz mobile phone-induced renal tissue damage, and CAPE, via its free radical scavenging and antioxidant properties, ameliorates oxidative renal damage. These results strongly suggest that CAPE exhibits a protective effect on mobile phone-induced and free radical mediated oxidative renal impairment in rats.     

Uric acid correlates to oxidation and inflammation in opposite directions in women

Abstract

Objective: To evaluate the association of uric acid (UA) levels with a panel of markers of oxidative stress and inflammation.

Methods: Plasma UA levels, along with a panel of oxidative stress and inflammatory markers, were measured in 755 Chinese women.

Results: Plasma UA levels were inversely associated with urinary levels of the oxidative stress marker F₂-isoprostanes and positively correlated to levels of inflammatory markers, such as C-reactive protein and some proinflammatory cytokines (tumor necrosis factor-α and interleukin-6) in blood as well as prostaglandin E₂ metabolites in urine.

Conclusions: Plasma UA levels correlate to oxidation and inflammation biomarkers in opposite directions in women.     

Urinary markers of nucleic acid oxidation in Danish overweight/obese children and youths

Urinary excretion of the RNA and DNA oxidation markers, 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in newly diagnosed adult type 2 diabetics are reported to be long-term predictors of mortality independent of conventional risk factors. In the current study, we investigated the relationships between urinary markers of nucleic acid oxidation concentrations and the degree of obesity and glucose metabolism in overweight compared to lean children. Forty-two (24 girls) overweight and 35 lean (19 girls) children and adolescents were recruited from the Registry of the Danish Childhood Obesity Biobank. Anthropometric measurements were collected at baseline and glucose metabolism was assessed by an oral glucose tolerance test. A urine sample was obtained during the test. Linear regression did not demonstrate any associations between the urinary markers and the degree of obesity or glucose metabolism in lean and obese children. However, sub-analyses adjusted for age, sex, and the degree of obesity showed positive associations between the 2 h glucose and the urinary markers, 8-oxoGuo (p?=?0.02, r²=?0.63) and 8-oxodG (p?=?0.046, r²=?0.48), and between the insulinogenic index and 8-oxoGuo (p?=?0.03, r^2?=^?0.60) in the 12 obese children exhibiting impaired glucose tolerance. Excretion of the urinary markers of nucleic acid oxidation and the degree of obesity or the glucose metabolism were not associated in this study. Nevertheless, obese children with impaired glucose tolerance seem to exhibiting an increased oxidative stress level, but due to the small sample size in this study, further investigations are required to elucidate this correlation.     

Temporal stability of urinary and plasma biomarkers of tobacco smoke exposure among cigarette smokers

Intraindividual variability of measurements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), nicotine, cotinine, and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) over time is uncertain. From 70 habitual smokers’ plasma and urine sampled bimonthly for a year we analysed plasma for NNAL, cotinine and PheT, and urine for NNAL, cotinine and nicotine. We estimated the intraclass correlation coefficients (ρ_I) for each measurement. Plasma and creatinine-corrected urinary NNAL were stable (ρ_I ≥70%); plasma PheT and plasma and urinary total cotinine were fairly stable (ρ_I ≥50%), but urinary nicotine ρ_I ≈ 40% was not. Except for nicotine, single measurements from plasma or urine adequately represent individual mean exposure over time.     

Influence of confounding factors on plasma Mid-Regional pro-Adrenomedullin and Mid-Regional pro-A-type Natriuretic Peptide concentrations in healthy individuals

Background: MRproADM and MRproANP can be used as diagnostic and prognostic markers in heart failure.

Aim: The objective was to identify confounding factors for the interpretation of plasma MRproADM and MRproANP concentrations.

Methods: A total of 518 healthy volunteers with a mean age of 60.84?±?7.41 years were analyzed. We evaluated the influence of demographic factors, renal function and echocardiographic indices on the candidate peptides.

Results: Multivariate analysis revealed that age (P?<?0.001), BMI (P?<?0.001) and eGFR (P?<?0.001) were independent predictors for MRproADM concentrations in healthy subjects. The independent predictors for MRproANP were age (P?<?0.001), female gender (P?<?0.001), heart rate (P?<?0.001) and eGFR (P?=?0.039).

Conclusion: The interpretation of both peptides is multifaceted due to confounders. Knowledge of these factors will further our understanding of how these peptides behave in health and in disease.