Acute Nutritional Stress Research Articles

Low feed intake at weaning reduces intestinal glutathione levels and promotes cysteine oxidation to taurine in pigs

Abstract Weaning stress in pigs is associated with low feed intake and poor nutrient utilization. Cysteine is a sulfur amino acid with key roles in pig production, but how cysteine metabolism and requirements are affected by weaning stress should be better defined. The objective of this study was to determine the collective impact of weaning and feed restriction on tissue cysteine metabolism. Pigs were weaned at 21-d age without access to feed (W; 6.90 ± 0.81 kg; n = 9; reflecting acute nutritional stress) or were not weaned and remained with the sow (NW; 6.81 ± 0.65 kg; n = 8). At euthanasia (23-d age), blood, bile, and liver, jejunum, and ileum tissues were collected. Plasma, bile, and tissue amino acid and amino thiol concentrations were analyzed by HPLC. Activity of glutamate cysteine ligase (GCL) and glutathione synthetase (GSS), enzymes needed for glutathione (GSH) production, and cysteine dioxygenase 1 (CDO1) were determined with activity assays followed by HPLC analysis of reaction products. Plasma (271 versus 192 ± 19 µmol/L; P < 0.001) and liver (417 versus 298 ± 33 nmol/g; P < 0.05) Cys concentrations were increased in W compared to NW pigs. Despite greater plasma Cys, jejunum and ileum Cys content were not affected by weaning (P > 0.10), whereas γ-glutamylcysteine (γ-GlyCys), the immediate precursor of GSH, declined in both jejunum (14.3 versus 9.7 ± 1.4 nmol/g; P < 0.01) and ileum (11.2 versus 6.4 ± 0.8 nmol/g; P < 0.001) in W pigs. Glutathione content was lower in the jejunum (1379 versus 1720 ± 70 nmol/g; P < 0.05) and ileum (1497 versus 1740 ± 74 nmol/g; P < 0.05) in W pigs. In the jejunum, GCL activity tended to be greater (0.56 versus 0.39 ± 0.07 nmol γ-GluCys • mg-1 • min-1; P < 0.10), whereas GSS activity tended to be lower (1.11 versus 1.38 ± 0.10 nmol GSH • mg-1 • min-1; P < 0.10) in W compared to NW pigs. In the ileum, the activities of GCL and GSS were not affected by weaning (P > 0.10). Although liver CDO1 activity was not different between groups (P < 0.10), liver taurine was greater in W compared to NW pigs (5115 versus 2336 ± 912 nmol/g; P = 0.001). Bile concentrations of Cys (1203 versus 279 ± 103 µmol/L; P < 0.001) and cysteinylglycine (203 versus 117 ± 33 µmol/L; P < 0.10), the direct product of GSH degradation, were greater in W compared to NW pigs. Collectively, these results suggest that systemic Cys is not effectively utilized for gut GSH production in newly weaned pigs and is instead oxidized to taurine and eliminated in bile.

Faecal cortisol levels in a wild Iberian red deer population are best explained by prior weather conditions

AbstractThe responsiveness of the hypothalamic–pituitary‐adrenocortical axis to stressors is crucial for wild animals to survive and adapt to environmental changes without compromising individual welfare. We analysed the influence of prior weather conditions, seasonality, the influx of ecotourism, and nutrition on stress levels in a wild population of red deer in a Mediterranean hunting reserve in the Pre‐Pyrenees, Spain. We used faecal cortisol metabolites as a proxy for physiological and psychological stress, and faecal triiodothyronine metabolites as an indicator of nutritional stress. Faecal analyses were chosen because it is a non‐invasive technique that does not alter an individual's behaviour, and it is easy to conduct in a wild population, something which presents added challenges due to the lack of a controlled environment. Our results indicate that prior weather conditions, rather than just seasonality, best explained the variations in these hormones within seasons and among years. On the contrary, the results showed that high levels of the cortisol hormone did not necessarily correspond to low levels of the triiodothyronine hormone. This could be a sign of acute nutritional stress in this population. Finally, we did not find an effect of the ecotourism influx on cortisol levels, suggesting that this deer population tolerates the presence of tourists.

262 Weaning-Induced Changes in Tissue Cysteine Utilization in Pigs

Abstract Weaning stress in pigs is associated with low and variable feed intake, poor nutrient utilization, and high feed costs. Cysteine is a sulfur amino acid with key roles in pig production, but how cysteine metabolism and requirements are affected by weaning stress should be better defined. The objective of this study was to determine weaning-induced changes in tissue cysteine metabolism. Pigs (initial BW = 7.23 ± 0.68 kg) were either weaned at 21 d age and euthanized at 23 d age (W; n = 9) or remained with the sow and euthanized at 23 d age (NW; n = 9). Weaned pigs were fasted to simulate acute nutritional stress but had free access to water. At euthanasia, blood, liver, jejunum, and ileum tissues were collected. Plasma and tissue amino acids and amino thiols were analyzed by HPLC. The abundance of proteins central to liver cysteine utilization were determined by immunoblot and enzyme activity assays. Glutathione (GSH) concentrations were less in jejunum (1,379 versus 1,720 ± 70 nmol/g; P < 0.01) and ileum (1,497 versus 1,740 ± 74 nmol/g; P < 0.05) in W than NW pigs. However, liver GSH concentration was not different between W and NW pigs (P > 0.10). While cysteine in jejunum and ileum were not affected by weaning (P > 0.10), γ-glutamylcysteine (γ-GC), the immediate precursor of GSH, declined in both jejunum (9.7 versus 14.3 ± 1.4 nmol/g; P < 0.01) and ileum (6.4 versus 11.2 ± 0.8 nmol/g; P < 0.001) in W compared with NW pigs. In liver, cysteine concentration was greater in W compared with NW pigs (417 versus 298 ± 33 nmol/g; P < 0.05). Although liver cysteine was greater in W pigs, liver activity of glutamate cysteine ligase (GCL; 2.49 versus 4.15 ± 0.25 nmol γ-GC/(mg protein•min); P < 0.001) and glutathione synthetase (GSS; 1.54 versus 1.92 nmol GSH/(mg protein•min); P < 0.001), enzymes needed for GSH production, were less in W than NW pigs. Immunoblot analysis of the catalytic subunit of GCL (GCLC; P < 0.05) and modifier subunit of GCL (GCLM; P = 0.057) were also lower in W compared with NW pigs, but GSS was not different between groups (P > 0.10). While plasma taurine was lower in W compared with NW pigs (64 versus 128 ± 7 µmol/L, P < 0.001), liver taurine was greater in W compared with NW pigs (4,726 versus 2,250 ± 471 nmol/g; P < 0.01). Despite an increase systemic and liver availability of cysteine, the rate-limiting amino acid for GSH synthesis, small intestine GSH concentrations decline. This suggests that systemic cysteine is not effectively utilized for gut GSH production in pigs after weaning.

Evolutionary responses of Drosophila melanogaster under chronic malnutrition.

Drosophila species have successfully spread and adapted to diverse climates across the globe. For D. melanogaster, rotting vegetative matter provides the primary substrate for mating and oviposition, and also acts as a nutritional resource for developing larvae and adult flies. The transitory nature of decaying vegetation exposes D. melanogaster to rapidly changing nutrient availability. As evidenced by their successful global spread, flies are capable of dealing with fluctuating nutritional reserves within their respective ecological niches. Therefore, D. melanogaster populations might contain standing genetic variation to support survival during periods of nutrient scarcity. The natural history and genetic tractability of D. melanogaster make the fly an ideal model for studies on the genetic basis of resistance to nutritional stress. We review artificial selection studies on nutritionally-deprived D. melanogaster and summarize the phenotypic outcomes of selected animals. Many of the reported evolved traits phenocopy mutants of the nutrient-sensing PI3K/Akt pathway. Given that the PI3K/Akt pathway is also responsive to acute nutritional stress, the PI3K/Akt pathway might underlie traits evolved under chronic nutritional deprivation. Future studies that directly test for the genetic mechanisms driving evolutionary responses to nutritional stress will take advantage of the ease in manipulating fly nutrient availability in the laboratory.

Acute nutritional stress during pregnancy affects placental efficiency, fetal growth and adult glucose homeostasis.

Exposure to maternal malnutrition impairs postnatal health. Acute nutritional stress is less clearly implicated in intrauterine programming.We studied the effects of stressing pregnant mothers on perinatal growth and adult glucose homeostasis. We compared one group (“stressed”, mothers fasted for 16 hours) with controls (“unstressed”). We found that fasting stress had adverse effects on the weight of the fetuses conceived (p<0.005) and the placental efficiency (p<0.001) in stressed compared to unstressed offspring. Placental weight was increased (p<0.001) presumably in compensation.Stress affected the glucose homeostasis of the offspring when they became adults (p<0.005) when analysed as individuals.We previously linked nutritional stress throughout pregnancy with a mitochondrial stress response. We modelled placenta with cultured human trophoblast cells (BeWos) and fetal tissues with mouse embryonic fibroblasts (MEFs). High throughput imaging showed that the mitochondria of both cell types underwent a similar sequence of changes in morphology, induced by nutritional stresses.The contrasting stress responses on fetal and placental weight were not captured by the cellular models. The stress of maternal fasting may be an important determinant of perinatal outcome in the mouse and might be relevant to nutritional stress in human pregnancy.

Effect of reducing milk production using a prolactin-release inhibitor or a glucocorticoid on metabolism and immune functions in cows subjected to acute nutritional stress.

When cows are unable to consume enough feed to support milk production, they often fall into severe negative energy balance. This leads to a weakened immune system and increases their susceptibility to infectious diseases. Reducing the milk production of cows subjected to acute nutritional stress decreases their energy deficit. The aim of this study was to compare the effects on metabolism and immune function of reducing milk production using quinagolide (a prolactin-release inhibitor) or dexamethasone in feed-restricted cows. A total of 23 cows in early/mid-lactation were fed for 5 d at 55.9% of their previous dry matter intake to subject them to acute nutritional stress. After 1 d of feed restriction and for 4 d afterward (d 2 to 5), cows received twice-daily i.m. injections of water (control group; n=8), 2mg of quinagolide (QN group; n=7), or water after a first injection of 20mg of dexamethasone (DEX group; n=8). Feed restriction decreased milk production, but the decrease was greater in the QN and DEX cows than in the control cows on d 2 and 3. As expected, feed restriction reduced the energy balance, but the reduction was lower in the QN cows than in the control cows. Feed restriction decreased plasma glucose concentration and increased plasma nonesterified fatty acid (NEFA) and β-hydroxybutyrate (BHB) concentrations. The QN cows had higher glucose concentration and lower BHB concentration than the control cows. The NEFA concentration was also lower in the QN cows than in the control cows on d 2. Dexamethasone injection induced transient hyperglycemia concomitant with a reduction in milk lactose concentration; it also decreased BHB concentration and decreased NEFA initially but increased it later. Feed restriction and quinagolide injections did not affect the blood concentration or activity of polymorphonuclear leukocytes (PMN), whereas dexamethasone injection increased PMN blood concentration but decreased the proportion of PMN capable of inducing oxidative burst. Incubation of peripheral blood mononuclear cells in serum harvested on d 2 of the restriction period reduced their ability to react to mitogen-induced proliferation, and injection of quinagolide or dexamethasone could not alleviate this effect. This experiment shows that prolactin-release inhibition could be an alternative to dexamethasone for reducing milk production and energy deficit in cows under acute nutritional stress, without disturbing immune function.

A nutrigenomic approach to detect nutritional stress from gene expression in blood samples drawn from Steller sea lions

Gene expression profiles are increasingly being used as biomarkers to detect the physiological responses of a number of species to disease, nutrition, and other stressors. However, little attention has been given to using gene expression to assess the stressors and physiological status of marine mammals. We sought to develop and validate a nutrigenomic approach to quantify nutritional stress in Steller sea lions (Eumetopias jubatus). We subjected 4 female Steller sea lions to 3 feeding regimes over 70-day trials (unrestricted food intake, acute nutritional stress, and chronic nutritional stress), and drew blood samples from each animal at the end of each feeding regime. We then extracted the RNA of white blood cells and measured the response of 8 genes known to react to diet restriction in terrestrial mammals. Overall, we found that the genomic response of Steller sea lions experiencing nutritional stress was consistent with how terrestrial mammals respond to dietary restrictions. Our nutritionally stressed sea lions down-regulated some cellular processes involved in immune response and oxidative stress, and up-regulated pro-inflammatory responses and metabolic processes. Nutrigenomics appears to be a promising means to monitor nutritional status and contribute to mitigation measures needed to assist in the recovery of Steller sea lions and other at-risk species of marine mammals.

School-Aged Children Felling Hunger at School Were at a Higher Risk for Thinness in Kersa District, Eastern Ethiopia: A Cross-Sectional Study

Background: Thinness in school children is a serious health condition with serious consequences for their development, health, and well-being. The objective of this study was to assess the magnitude and associated factors of thinness among school age children in Eastern Ethiopia. Methods: A cross sectional study was conducted among school age children in Eastern Ethiopia. A child was identified as thin if Body Mass Index for -age Z-score is below minus two standard deviations. Result: Thinness was observed among 206 of 1768 school children [11.6%; 95% CI; CI 10.1- 13.1); about 2% had very low BMI for age. Children in the age group 13-14 years had 1.62 times the risk of thinness compared to children aged 5-9 years (AOR=1.62; 95% CI: 1.06, 2.39). The risk of thinness among children reported felling hunger at school was 1.55 times as high as the risk of thinness compared to children who did not report felling hunger at school (AOR=1.55; 95% CI: 1.07, 2.23). Children from families who did not have latrine had 1.53 times the risk of thinness compared to children from families who had latrine (AOR=1.53; 95% CI: 1.09, 2.13). On the other hand, children born to older mother (age ≥ 35 year) had 30% less risk of being thin compared to children born from younger mothers (AOR 0.70; (95% CI), 0.51-0.98). Conclusion: This study provided evidence that over 10% of school children were under acute nutritional stress in the form of thinness. Thus, integration of nutrition interventions into a comprehensive school health program including free school meals can potentially benefit those children.

Herp depletion protects from protein aggregation by up-regulating autophagy

Herp is an endoplasmic reticulum (ER) stress inducible protein that participates in the ER-associated protein degradation (ERAD) pathway. However, the contribution of Herp to other protein degradation pathways like autophagy and its connection to other types of stress responses remain unknown. Here we report that Herp regulates autophagy to clear poly-ubiquitin (poly-Ub) protein aggregates. Proteasome inhibition and glucose starvation (GS) led to a high level of poly-Ub protein aggregation that was drastically reduced by stably knocking down Herp (shHerp cells). The enhanced removal of poly-Ub inclusions protected cells from death caused by glucose starvation. Under basal conditions and increasingly after stress, higher LC3-II levels and GFP-LC3 puncta were observed in shHerp cells compared to control cells. Herp knockout cells displayed basal up-regulation of two essential autophagy regulators—Atg5 and Beclin-1, leading to increased autophagic flux. Beclin-1 up-regulation was due to a reduction in Hrd1 dependent proteasomal degradation, and not at transcriptional level. The consequent higher autophagic flux was necessary for the clearance of aggregates and for cell survival. We conclude that Herp operates as a relevant factor in the defense against glucose starvation by modulating autophagy levels. These data may have important implications due to the known up-regulation of Herp in pathological states such as brain and heart ischemia, both conditions associated to acute nutritional stress.

Adipose Tissue Lymphocytes and Macrophages in Obesity and Insulin Resistance

Obesity, and more specifically accumulation of adipose tissue in the visceral and subcutaneous abdominal locations, is a major risk factor for the development of cardiovascular pathologies including hypertension and atherosclerosis, as well as metabolic disorders such as type 2 diabetes. During recent years, “metaflammation” or metabolically-triggered inflammation1 has emerged as a key process involved in the clustering of those conditions. Although several metabolically active organs such as the liver, muscle, and, recently, the intestine2 certainly play major roles, the white adipose tissue appears as a central and primary player as both a source and site of inflammation. Accumulation of adipose tissue macrophages (ATMs) has been well-described in obese conditions in mice and humans.3–5 Moreover, the ATM proinflammatory phenotype has been linked to the development of insulin resistance in mice,4 although the exact nature of the proinflammatory myeloid cells, ie, macrophages or dentritic cells, remains to be determined.6 Nevertheless, the causal link between inflammation and insulin resistance was further strengthened by the specific knock-out of the inflammation coordinator IkappaB kinase beta of myeloid cells, which gave protection against insulin resistance.7 The study of Kintscher and al in this issue8 extends those original observations to cells of adaptative immunity. The authors suggest that the accumulation of T-lymphocytes, assessed mainly through gene expression analyses and immunohistochemistry, occurs in the perigonadal adipose tissue of mice on …

Larval Life History Responses to Food Deprivation in Three Species of Predatory Lady Beetles (Coleoptera: Coccinellidae)

We studied life history responses of larvae of three coccinellid species, Coleomegilla maculata (DeGeer), Hippodamia convergens Guerin-Meneville, and Harmonia axyridis (Pallas), when deprived of food for different periods of time during the fourth stadium. The coccinellid species did not differ in starvation resistance when larvae were starved throughout the stadium; however, for larvae fed only on day 1 of the stadium, H. convergens had the highest starvation resistance, followed by H. axyridis and then C. maculata. Percentage weight loss of larvae was affected by food deprivation period and coccinellid species. Both C. maculata and H. axyridis lost significantly more weight than H. convergens when starved throughout the fourth stadium. When deprived of food for 4 d of the stadium, C. maculata lost a higher percentage of initial body weight than H. axyridis. Percentage weight loss of H. convergens did not differ from that of C. maculata or H. axyridis. The weight of fourth instars and adults declined in an accelerating pattern as food deprivation period increased. However, food deprivation period had no significant effect on pupal development time for any of the three species or on larval development time for C. maculata and H. convergens. The increase in H. axyridis larval development time as a result of an increase in food deprivation period was curvilinear. Based on this laboratory study, it would seem that H. convergens is better able to cope with acute nutritional stress than either C. maculata or H. axyridis.

Larval Life History Responses to Food Deprivation in Three Species of Predatory Lady Beetles (Coleoptera: Coccinellidae)

We studied life history responses of larvae of three coccinellid species, Coleomegilla maculata (DeGeer), Hippodamia convergens Guerin-Meneville, and Harmonia axyridis (Pallas), when deprived of food for different periods of time during the fourth stadium. The coccinellid species did not differ in starvation resistance when larvae were starved throughout the stadium; however, for larvae fed only on day 1 of the stadium, H. convergens had the highest starvation resistance, followed by H. axyridis and then C. maculata. Percentage weight loss of larvae was affected by food deprivation period and coccinellid species. Both C. maculata and H. axyridis lost significantly more weight than H. convergens when starved throughout the fourth stadium. When deprived of food for 4 d of the stadium, C. maculata lost a higher percentage of initial body weight than H. axyridis. Percentage weight loss of H. convergens did not differ from that of C. maculata or H. axyridis. The weight of fourth instars and adults declined in an accelerating pattern as food deprivation period increased. However, food deprivation period had no significant effect on pupal development time for any of the three species or on larval development time for C. maculata and H. convergens. The increase in H. axyridis larval development time as a result of an increase in food deprivation period was curvilinear. Based on this laboratory study, it would seem that H. convergens is better able to cope with acute nutritional stress than either C. maculata or H. axyridis.

P2-62 Effects of acute nutritional stress during the pre-implantation/implantation stage of gestation on fetal origin of adult disorders in the mouse

P2-62 Effects of acute nutritional stress during the pre-implantation/implantation stage of gestation on fetal origin of adult disorders in the mouse

Urinary cortisol and muscle mass in Turkana men

To determine the role of cortisol in modulating the effects of energetics on muscle mass in a subsistence society, measures of cortisol and body composition were obtained from a sample of Turkana men. Subjects were 63 settled and 69 nomadic men, ages 24 and older. Urinary cortisol was determined along with measures of muscle mass. Cortisol/creatinine ratio was higher among nomadic men (57.8 +/- 56.8 vs. 34.5 +/- 44.6; P < 0.001). Controlled for age, urinary cortisol was inversely related to arm muscle plus bone area (MPBA) among the nomadic (beta = -0.28; P = 0.04), but not the settled (beta = -0.04; P = 0.78) sample. Urinary cortisol was not related to any other measures of body composition. These results suggest that even in chronically undernourished populations, cortisol may be elevated primarily under conditions of acute nutritional stress. However, the catabolic effects of cortisol on muscle in our results may be confounded by other energetic factors, including energy availability.

The decline of Steller sea lions Eumetopias jubatus in Alaska: a review of the nutritional stress hypothesis

ABSTRACT 1. The decline of Steller sea lions Eumetopias jubatus in the Gulf of Alaska and Aleutian Islands between the late 1970s and 1990s may have been related to reduced availability of suitable prey. Many studies have shown that pinnipeds and other mammals suffering from nutritional stress typically exhibit reduced body size, reduced productivity, high mortality of pups and juveniles, altered blood chemistry and specific behavioural modifications. 2. Morphometric measurements of Steller sea lions through the 1970s and 1980s in Alaska indicate reduced body size. Reduced numbers of pups born and an apparent increase in juvenile mortality rates also appear to be nutritionally based. Blood chemistry analyses have further shown that Steller sea lions in the Gulf of Alaska and Aleutian Islands area exhibited signs of an acute phase reaction, or immune reaction, in response to unidentified physical and/or environmental stress. Behavioural studies during the 1990s have not noted any changes that are indicative of an overall shortage in the quantity of prey available to lactating female sea lions. 3. The data collected in Alaska are consistent with the hypothesis that Steller sea lions in the declining regions were nutritionally compromised because of the relative quality of prey available to them (chronic nutritional stress), rather than because of the overall quantity of fish per se (acute nutritional stress). This is further supported by captive studies that indicate the overall quality of prey that has been available to Steller sea lions in the declining population could compromise the health of Steller sea lions and hinder their recovery.

Correlates of dietary intake of lactating women in South Turkana.

Previous studies of nutrition among nomadic pastoralists in South Turkana, Kenya, have suggested that social mechanisms may be used to buffer vulnerable members of the group from acute nutritional stress. During 1989-1990, 24-hour dietary recall data were collected from 101 lactating women in Ngisonyoka, Turkana. Multivariate regression was then used to quantify the effects of environmental, socioeconomic, and demographic variables on dietary intake. The location of individual herding camps had the greatest impact on maternal diet. After the site of camps, rainfall patterns, the socioeconomic ranking of the herding unit, and the age and rank of wives were the most significant predictors of maternal food consumption. The results suggest that some women in the study were buffered from moderate seasonal nutritional stress by the practice of food-sharing among members of their social network, as well as by voluntary reduction in dietary intake by other women. Maternal dietary intake differed both qualitatively and quantitatively between herding camps of higher and lower socioeconomic ranking. © 1994 Wiley-Liss, Inc.

The effect of variations in vitamin C intake on the cellular immune response of guinea pigs

The uptake of tritiated thymidine by isolated peripheral blood lymphocytes obtained from male guinea pigs immunized with bovine serum albumin was studied in animals maintained on various amounts of Vitamin C for 28 days. Animals were pair-fed on ascorbate-free diet and were supplemented intraperitoneally with 0, 25, or 250 mg Na-ascorbate per day. Scorbutic animals lost weight rapidly during the final 2 experimental weeks. Their daily food intake averaged only 4 g/day during the last week; thus, pair-fed ascorbate-supplemented groups were also subjected to acute nutritional stress. Lymphocytes from guinea pigs receiving 250 mg Na-ascorbate per day incorporated in vitro the highest amounts of tritiated thymidine both in the absence of nonspecific mitogen and in the presence of concanavalin A or phytohemagglutinin. Responses to lipopolysaccharide were not conclusive. Total circulating white cells counts and relative numbers of T and B lymphocytes were assessed in a second study made under identical constraints. In scorbutic animals the percentage of B lymphocytes increased and that of T lymphocytes decreased continuously over the 4-week period. The opposite effect was observed in vitamin C-supplemented animals. These studies suggest that very high doses of ascorbate support elevated mitotic activity after 4 weeks of much reduced food intake.

Influence of Acute Nutritional Stress on Polyribosomes and Protein Synthesis in Brain and Liver of Young Rats

Polyribosomal patterns and protein synthesis of brain and liver were studied in 10-day-old suckling rats fasted for 6 to 12 hours and in 35-day-old post-weanling rats fasted for 3 days or force-fed a threonine-devoid diet for 3 days. Fasting in either the 10- or 35-day-old rats resulted in disaggregation of polyribosomes and a decrease in protein synthesis of liver as measured in vitro whereas polyribosomes and in vitro assay of protein synthesis of brain were unaffected. Force-feeding of a threonine-devoid diet for 3 days, which has been shown to induce kwashiorkor-like lesions in pancreas, salivary glands and liver along with enhanced hepatic protein synthesis, had no effect on brain polyribosomes and on brain protein synthesis when measured in vivo or in vitro.