AMP-Activated Protein Kinase Regulates Oxidative Metabolism in Caenorhabditis elegans through the NHR-49 and MDT-15 Transcriptional Regulators

Cellular energy regulation relies on complex signaling pathways that respond to fuel availability and metabolic demands. Dysregulation of these networks is implicated in the development of human metabolic diseases such as obesity and metabolic syndrome. In Caenorhabditis elegans the AMP-activated protein kinase, AAK, has been associated with longevity and stress resistance; nevertheless its precise role in energy metabolism remains elusive. In the present study, we find an evolutionary conserved role of AAK in oxidative metabolism. Similar to mammals, AAK is activated by AICAR and metformin and leads to increased glycolytic and oxidative metabolic fluxes evidenced by an increase in lactate levels and mitochondrial oxygen consumption and a decrease in total fatty acids and lipid storage, whereas augmented glucose availability has the opposite effects. We found that these changes were largely dependent on the catalytic subunit AAK-2, since the aak-2 null strain lost the observed metabolic actions. Further results demonstrate that the effects due to AAK activation are associated to SBP-1 and NHR-49 transcriptional factors and MDT-15 transcriptional co-activator, suggesting a regulatory pathway that controls oxidative metabolism. Our findings establish C. elegans as a tractable model system to dissect the relationship between distinct molecules that play a critical role in the regulation of energy metabolism in human metabolic diseases.


Introduction
Cellular energy balance is maintained by several cell signaling networks that regulate the uptake, storage and utilization of nutrients. Defects in maintaining energy homeostasis may lead to metabolic diseases, such as obesity, metabolic syndrome and type II diabetes [1]. Since many key signaling mechanisms in mammals appear to be conserved in the nematode Caenorhabditis elegans, this free-living worm has been used as an experimental model to explore the

Time-dependent phosphorylation of AAK by AICAR and metformin exposure in C. elegans
To determine the role of AAK in the regulation of energy metabolism in C. elegans, we first assessed whether it is susceptible to phosphorylation (activation) by two standard activators of AMPK (AICAR or metformin) [25]. Adult wild-type N2 nematodes were exposed to AICAR or metformin at different times (2, 12, 24, 36 and 48 hours). We found that AAK phosphorylation increased in a time-dependent manner with both activators, being earlier and more pronounced with AICAR than with metformin (12 versus 24 h, respectively, Fig 1A and 1B). Hence, 12 and 24 h with AICAR or metformin were used in all subsequent experiments to determine the effect of AAK phosphorylation upon oxidative metabolism. To exclude that the observed surge in AAK phosphorylation during exposure to these activators was due to increased expression of the AAK protein, we measured mRNA expression of the aak-1 and aak-2 genes in wild-type N2 animals exposed to AICAR or metformin. Our results showed no differences in the expression levels of these genes (Fig 1C). Even more, in the AAK-2 null mutant (ok524) no changes in gene expression of the remaining subunit AAK-1 were observed ( Fig 1C). These results demonstrate that both AICAR and metformin increased AAK phosphorylation in C. elegans in a time-dependent manner without altering protein expression.

Energy metabolism is changed by AAK phosphorylation in C. elegans
Because AMPK activation regulates metabolic flux of nutrients such as carbohydrates and lipids [26], we determined the effect of AAK activation on some energy metabolic markers, such as azide-sensitive oxygen consumption (which is an index of mitochondrial respiration), lactate production and total triglycerides content. We found that the mitochondrial oxygen-consumption rates (Fig 2A) and concentration of lactate ( Fig 2B) significantly increased, whereas triacylglycerol (TAG) content decreased (Fig 2C) in N2 wild type nematodes with their AAK activated by AICAR or metformin. To explore whether these changes depended on the AAK-2 subunit, we exposed a null mutant strain of AAK-2 [12] to the AAK activators AICAR or metformin. We found no changes on the physiological outcomes in the aak-2 mutant strain Fig 1. AICAR and metformin increased AAK phosphorylation in a time-dependent manner. AAK phosphorylation at Thr243 (equivalent to Thr172 of mammals) was detected using an anti-pAMPK antibody in N2 adult nematodes treated with A) 1 mM AICAR or B) 50 mM metformin. In both cases, the top panel shows a representative Western blot of phosphorylated AAK (pAAK), where the intensity of the bands represents the phosphorylation level, corresponding to kinase activation. C) Expression levels of aak-2 and aak-1 by qRT-PCR in N2 and aak-2/ok524 strains exposed to 1 mM AICAR for 12 h or 50mM Metformin for 24 h. The graphs show the mean ± SEM of three independent experiments *p<0.05, **p<0.01 vs. 0 h, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism. (Fig 2A-2C). The decreased TAG concentrations could be in line with the increased oxidative metabolism due to AAK activation, as reflected by higher respiration, suggesting that AAK-2 is necessary to promote oxidative metabolism by AICAR and metformin in C. elegans. AAK activation by AICAR and metformin enhances oxidative metabolism in C. elegans. N2 (white box) and aak-2/ok524 (black box) nematode strains were grown to the adult stage and exposed to 1 mM AICAR for 12 h or 50 mM metformin for 24 h. A) Mitochondrial and non-mitochondrial oxygen consumption. B) Lactate concentration and C) Total triglyceride content. Each graph shows the mean ± SEM of three independent experiments. The aak-2 mutant groups showed no change between them. *p<0.05, **p<0.01, ***p<0.001 vs. untreated group, one-way ANOVA with Bonferroni's post hoc test, using GraphPad Prism.

AAK activation modifies fatty acid composition in C. elegans
Since reduction of TAG content seems to depend on AAK activation and because TAGs are synthesized depending on fatty acid availability [27], we hypothesized that its activation may change total fatty-acid composition in the worm. Thus, we measured the amount of individual fatty acids in wild-type N2 nematodes after AAK activation and grouped them into saturated fatty acids (SFA), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) (Fig 3A and 3B). Although there were no statistically significant changes in the individual fatty acids; palmitic (C16:0), palmitoleic (C16:1n7), 7-palmitoleic acid (C16:1n9), vaccenic (C18:1n7) and oleic acid (18:1n9), levels of this fatty acids showed a tendency to augment, most likely as a result of the reduction of TAG synthesis induced by AAK activation. In the other hand, di-homo-γ-linolenic (C20:3n6), arachidonic (C20:4n6), omega-3 arachidonic (C20:4n3) and eicosapentaenoic acids (C20:5n3) were reduced upon AAK activation ( Fig 3A). Nevertheless, when we analyzed fatty acids by groups, PUFAs showed a statistically significant decrease when AAK was activated. Assuming that this group is the end point of the fatty acid synthesis pathway, this may reveal the effect of AAK in this metabolic route ( Fig 3B).

Effects of high glucose availability are reversed by AAK activation in C. elegans
Metformin is a first-line drug for treating type 2 diabetes; its proposed mechanism of action involves the AMPK activation [32]. In mammals, chronic exposure to high glucose negatively regulates this kinase [33,34]. To evaluate whether similar effects occur in C. elegans, we exposed wild-type N2 nematodes to a high glucose concentration (100 mM) in the presence or absence of metformin (50 mM, 24 h). We observed that basal AAK phosphorylation decreased upon glucose administration with respect to control animals, whereas treatment with both glucose and metformin significantly increased AAK phosphorylation even more than control nematodes ( Fig 7A). In accordance with a previous report [17], we also found that high glucose provision induced TAG accumulation and decreased the respiratory rate in C. elegans (Fig 7B and  7C). Since we demonstrated that AAK activation decreased TAG content in N2 wild type worms, it is plausible that AAK activation also modifies TAG accumulation plus oxidative metabolism and prevents deleterious effects when the worms are exposed to a high glucose concentration. As was expected, AAK activation returned TAG content and oxygen consumption to control levels, even in presence of higher glucose availability (Fig 7B and 7C). Altogether, our results suggest that metformin could reverse the effects generated by high glucose availability in an AAK-dependent manner, since such changes were not observed in the aak-2 mutant strain exposed to high glucose or high glucose plus metformin (Fig 7D and 7E).

Discussion
Here, we demonstrate that AAK, the C. elegans AMP-activated protein kinase, can be activated pharmacologically and this modifies energy metabolism by increasing oxidative flux, as indicated by the increase in lactate levels and oxygen consumption. AAK activation by phosphorylation promotes lipid mobilization and decreases PUFAs synthesis. We propose that AAK, NHR-49 and MDT-15 form a regulatory system that coordinates systemic adaptation to energy changes, which in turn, ensures maintenance of homeostasis and health. These data are consistent with the widespread role of AMPK in metabolic control, suggesting that its activation is executed via a similar mechanism from nematodes to humans.

AAK activation by AICAR and metformin promotes oxidative metabolism in C. elegans
The fact that the AMPK energy sensor is evolutionarily conserved [10,35], suggests that it may also play an important role in controlling energy metabolism in C. elegans. To test this idea, we first activated AAK by AICAR or metformin exposure. The concentrations of AICAR and metformin used in our experiments were sufficient to increase AAK activation over the time course evaluated, being faster and more pronounced by AICAR. This fact may reflect a positive allosteric effect of the ZMP, the phosphorylated form of AICAR, similar to AMP, in the worm kinase, which takes place with maximal phosphorylation at 12 h; whereas with metformin this effect was reached until 24 hours of exposure, consistent with the accepted consensus that metformin indirectly activates AMPK [32]. Nevertheless, we cannot exclude the possibility that AICAR has a different potential to activate AAK in comparison with metformin, which would need further experimental exploration.
It is well known that AMPK activation reestablishes cellular energy in part by activation of oxidative metabolism [9]. Consistently, we observed an increase in lactate production and mitochondrial oxygen consumption, but a decrease in lipid storage after AAK activation ( Fig  2); suggesting an increased glycolytic and lipolytic flux, as well as augmented β-oxidation, which could explain the observed higher respiration rates and hence, the elevated oxidative metabolic flux. Under AAK activation both, glycolysis and TCA cycle are enhanced at independent ways; lactate production may be increased by effect on rate-limiting glycolytic enzymes hexokinase and phosphofructokinase I and II [36,37], whereas TCA cycle and oxidative phosphorylation may be stimulated by augmenting beta-oxidation that directly supplies acetyl-CoA and reducing equivalents, which in turn reduces pyruvate flux to TCA cycle; in order for glycolysis to proceed, accumulated pyruvate will then rapidly diverge to lactate [36]. Stimulation of the phosphofructokinase I has been shown to increase lactate levels even if aerobic glucose  metabolism occurs [38]. The results agree with a recent report where proteomic profiles showed that metformin-treated nematodes have up-regulated expression of several proteins from glycolysis and the tricarboxylic acid cycle (TCA) and corresponding increased respiration rates [39]. Augmented oxygen consumption by metformin treatment a priori seems inconsistent with the general idea that metformin inhibits complex I of the mitochondrial electron transport chain and hence decreases respiration [40]. But our data as well as previous reports [39,40], indicate that increased carbohydrate and fatty acid oxidation produce mediators that accelerate the TCA cycle to activate mitochondrial respiration. Indeed, metformin inhibition of complex I is an acute process, whereas the observed result in the worm seems a long term effect. It is noteworthy that fatty acid oxidation cannot occur when mitochondrial respiration is interrupted; supporting the fact that metformin treatment may result in up-regulation of several catabolic processes as a consequence of sustained AAK activation in C. elegans.
In C. elegans, the orthologs of the AMPK catalytic subunits α1 and α2 are encoded by the aak-1 and aak-2 genes [12]. It has been proposed that AAK-2 participates in longevity, stress resistance and some effects of dietary restriction, whereas mutation of AAK-1 does not cause obvious abnormalities [12,13,15,21,41,42]; suggesting that AAK-2 is the subunit responsible for its kinase activity. Nevertheless, the effects of AAK-2 activation on energetic metabolism in C. elegans are still unknown. In this sense, our data suggest that AAK-2 primarily modulates energy metabolism, in view of the fact that its activation modifies several metabolic markers such as lactate production, TAGs and oxygen consumption. Accordingly, it was plausible to expect that aak-2 deletion declines the mitochondrial activity reflected by a reduced-oxygen consumption, which in turn relieves inhibition of glycolytic flux and promotes lactate accumulation, even more than in AAK activated state. Consistently, in this paper we found that in animals carrying aak-2 deletion, lactate production is more increased than in wild type animals with the AAK activated (Fig 2B), while mitochondrial oxygen consumption rate is decreased (Fig 2A).
Moreover, in nematodes lacking the AAK-2 protein (aak-2 null, ok524) metformin or AICAR failed to change the mentioned catabolic fluxes (Fig 2), supporting that the fuel-conserving role of AMPK is evolutionarily conserved.

Metabolic changes triggered by AAK are dependent on NHR-49, MDT-15
Because AAK activation diminishes fatty-acid storage, it is possible that this regulation might be mediated by transcription factors as occurs in mammals. We observed that the NHR-49, MDT-15 and SBP-1 transcription factors are involved in the effects produced by AAK activation, such as reduced total TAGs and PUFAs (Figs 2C and 3A), suggesting a possible link between AAK activity and these metabolic molecules.
The C. elegans functional orthologue of mammalian PPARα, NHR-49, is a key regulator of fat mobilization. This factor couples β-oxidation and desaturation of lipids by regulating several enzymes such as ACS-2 (acyl-CoA synthetase), CPT-5 (carnitine palmitoyltransferase), ECH-1 (enoyl-CoA hydratase) and those with Δ9-desaturase activities (FAT-5, FAT-6 and FAT-7) [22,23] that can stimulate mitochondrial respiration. We found that AAK activation increased gene expression of nhr-49, fat-7 and acs-2 (Fig 4A, 4D and 4G), as well as augmenting Δ9-desaturase activities ( Fig 3D); suggesting that the observed reduction of TAGs and increase of oxygen consumption were probably mediated by NHR-49, since no changes were observed in the mutant nhr-49 strain treated with AICAR (Fig 6A and 6C). Moreover, in the mutant lacking AAK-2, expression of acs-2 was severely depressed (Fig 4G), indicating the participation of this component in the reducing effect of AAK activation on TAG content ( Fig  6C), as well as the link between AAK and NHR-49 to modulate lipid oxidation through ACS-2. These findings are in agreement with the function of mammalian PPARα, which driven by AMPK is able to modulate enzymes involved in lipid catabolism [43,44].
Like NHR-49, MDT-15 is also vital for metabolic control by activating gene expression of acdh-1/2 and fat-2, which encode a short-chain acyl-CoA dehydrogenase and a Δ12-fatty acyl desaturase respectively, related to β-oxidation and lipid synthesis [8]. FAT-2 desaturase controls the conversion of MUFAs to PUFAs by catalyzing the first limiting step of PUFAs synthesis [29]. Our data showed diminished mdt-15 and fat-2 gene expression (Fig 4B and 4E), as well as reduced FAT-2 desaturase activity ( Fig 3D) and lower amounts of 18:2n6 and C20 PUFAs (Fig 3A and 3B) after AAK activation. By contrast, SFA such as palmitic and stearic acids remained with slight or no changes (Fig 3A). This can be explained at least in part, because more than 90% of palmitic acid is provided to C. elegans from the bacteria in the diet [4,45]. On the other side, enhanced beta-oxidation by activation of AAK may contribute to keep palmitic acid and other SFA at steady state levels.
However, no changes were observed in the mdt-15 null mutant upon AAK activation (Figs 5 and 6); suggesting that MDT-15, as NHR-49, is also involved in fatty acid desaturation and β-oxidation to maintain lipid homeostasis. MDT-15 in C. elegans has been reported to serve the same function as the mammalian PGC-1α [8]. This gene is up regulated by AMPK [46,47], but we did not observe an increase in MDT-15 expression under our conditions of AAK activation ( Fig 4B). Nevertheless, the intriguing decrease of MDT-15 that we observed could ensure the appropriate gene expression to cooperate with the metabolic effects orchestrated by AAK activation.
Moreover, as in mammals, lipid synthesis in C. elegans is also mediated by the SREBP homolog SBP-1 by augmenting the transcription of genes coding for lipogenic enzymes, such as elo-2, acc-1 and fasn-1 that drive fat accumulation and adipogenesis [6,48]. Although sbp-1 expression did not change by AAK activation; its target genes, fasn-1 and elo-2, as well as the estimated activity of elongase ELO-2 significantly decreased (Fig 4F and 4I). ELO-2 is responsible for the elongation of palmitic acid and PUFA biosynthesis [28]. Consistently, we observed a slight increase in palmitate and a clear diminution of PUFAs (Fig 3A and 3B). As mentioned before, palmitic acid in C. elegans comes primary from the diet [4,45]. By contrast, PUFA are not available in the main source of food for C. elegans when cultured in the laboratory (E. Coli OP50) then, PUFA found in the nematode come from de novo synthesis [4,45]. Thus, the high availability of palmitic acid probably maintains its level near to the steady state, in part because of its participation in the biosynthesis of MUFA mediated by FAT-6/7, although FAT-2 is hindered. As well, AMPK-stimulated β-oxidation may contribute to the balance of palmitic acid and other SFA levels. Since the Δ12 desaturase is the first step in the synthesis of PUFA, strains lacking this activity would be expected to be devoid of PUFA. Indeed, our data showed diminished fat-2 gene expression (Fig 4E), as well as reduced FAT-2 desaturase activity (Fig 3D) after AAK activation. It is conceivable that if the rate-limiting step for the synthesis of PUFA is decreased, no changes will be reflected in MUFAs, even if the activity of FAT-7 increases.
Although mammalian liver SREBP is inhibited by phosphorylation upon AMPK activation [49], this posttranslational modification has not yet been demonstrated in C. elegans; thus, our results suggest that a similar event may occur in the worm by AAK activation. Besides, it was shown that SREBP1c regulates expression of the enzyme that elongates C18-20 for PUFA biosynthesis (Elovl5) [50]. Interestingly, miRNAs, emerging as important metabolic regulators, in C. elegans have been implicated in the control of lipid metabolism. In fact, it has been shown that miR-786 directly represses elo-2 expression resulting in significantly elevated palmitate levels [51]. Although to our knowledge no data for fasn-1 is available, we cannot exclude the possibility that it is also regulated in a similar fashion, because miR-122 affects the expression of the mammalian fasn-1 orthologue, fatty acid synthase (FAS) [52]. Taken together, the data enables us to suggest that similar miRNA mechanisms might operate in our model. Thus, the apparent routing between MDT-15, NHR-49 and likely SBP-1, might explain the increased oxidative metabolism, as a result of the enhanced glycolytic and lipolytic rates that feed β-oxidation, which could operate as substrate sources for the mitochondrial respiratory chain. Additionally, determining changes of fatty acid profiles in AAK activator-treated nhr-49/ok2165, mdt-15/tm2182 and sbp-1/ok2363 mutant strains, would be interesting for deep knowledge about the AAK axis in the metabolic control in C. elegans.

AAK activation by metformin reverses the effects evoked by high glucose diet in C. elegans
Glucose is an energy molecule and its systemic levels are actively regulated, such that disruption of its metabolism can lead to human diseases like obesity and type 2 diabetes. In this sense, a high-glucose media has deleterious effects on C. elegans, suggesting altered metabolic homeostasis [53,54]. In our study, we observed that nematodes exposed to high glucose exhibited increased TAG content, diminished mitochondrial oxygen consumption and decreased AAK phosphorylation. Given the important role of AMPK as a "fuel gauge" [9,10] plus a strong link between low AMPK activation and a state of over-nutrition [55], it is plausible that a dysfunction in the AAK signaling pathway by a carbohydrate-rich diet may result in systemic perturbations that contribute to impaired mitochondrial metabolism, as previously suggested [17]. Following the enhanced effect of metformin on the oxidative fluxes in C. elegans, we wondered whether this drug was able to reverse the metabolic disruption evoked by high glucose exposure in C. elegans. Strikingly, we showed that in wild-type nematodes metformin prevented the effects generated by excess glucose intake, restoring the metabolic markers to control levels; similar to what occurs in obese and diabetic patients, where chronic metformin treatment decreases glucose and lipid levels in plasma [56]. In contrast, growing aak-2 mutant nematodes on high glucose and metformin did not produce any metabolic changes (Fig 7D and 7E), implying that AAK is a cellular metabolic checkpoint responding to an altered energy status, such as high glucose feeding.
In sum, we show the role of the energy sensor AAK in the metabolic control of C. elegans, likely through the AAK/NHR-49/MDT-15 axis. This axis is an integral part of a regulatory mechanism that coordinates systemic adaptation to energy challenges, which ensures and maintains energy homeostasis and hence promotes a healthy state. In addition, this work provides evidence that supports a relationship between nutritional perturbations and transcriptional factors that trigger mechanisms to restore energy homeostasis in an AAK-dependent fashion in C elegans. Fatty acid and carbohydrate metabolism are complex processes that are highly associated with pathologies such as diabetes or obesity. Additionally, many gaps in knowledge still exist, partly due to the lack of in vivo research concerning energy metabolism regulation. Thus, C. elegans is potentially an exciting screening model of metabolic signaling pathways to discover new therapeutic targets in pathologies associated with energy alterations.

Determination of AAK activation
AAK phosphorylation (activation) was evaluated by Western blot. The nematodes were homogenized mechanically using a mortar and pestle with protein lysis buffer [50 mM HEPES, 50 mM KCl, 1 mM EDTA, 1 mM EGTA, 5 mM β-glycerol phosphate, 0.1% Triton X-100, protease inhibitor cocktail (Complete, Roche), 50 mM NaF, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate and 0.2 mM PMSF], collected and centrifuged (10,000 g, 10 min, 4°C). The protein concentration was quantified using the Bradford method [59]. 30 μg of protein was subjected to SDS-PAGE and transferred to PVDF membranes. pAAK was identified using an anti-phospho-AMPKα antibody (Cell Signaling Technology). The signal was quantified using chemiluminescent detection and Quantity One 1-D Analysis v4.6.5 image processing software (Bio-Rad). An anti-actin antibody was used as the loading control.

Lactate and triglyceride quantification
Lactate concentration was determined using the biochemical end point method described previously (based on the Lactate Dehydrogenase assay) [60,61]. Briefly, the nematodes were mechanically homogenized in fresh HClO 4 3%/EDTA 20 mM, the supernatant resulting from centrifugation was neutralized in KOH 4N and used immediately to measure lactate. Although values for lactate are reported at nmol/mg protein, we found that 1 mM of lactate is approximately equivalent to 40.4 nmol of lactate/mg protein in one day of adulthood N2 nematodes. Which corresponds to values already reported by others [62].
Triglycerides were essentially quantified as described previously [63]. Briefly, the nematodes were homogenized mechanically in protein lysis buffer and then heated to 90°C for 5 min, followed by vortexing and centrifugation (10,000 g, 10 min, 20°C). The concentration was determined using a TAG assay kit (Sigma-Aldrich) by lipase enzymatic hydrolysis of the triglycerides to glycerol and free fatty acids. Metabolite concentrations were normalized to the total protein concentration of each sample as described before.

Oxygen consumption rate
The oxygen consumption rate was determined essentially as described previously [61], using a Clark-type oxygen electrode and an YSI 5300A Biological Oxygen Monitor. The living nematodes were collected, washed to remove excess bacteria and re-suspended in 50 μl of M9 buffer. The slopes were recorded for at least 10 minutes at 24°C [64] on a Bio-Rad 1325 Econo-Recorder. The O 2 solubility value used was 202.94 nmolO 2 /ml at 24°C, 585mmHg and 77% saturation, corresponding to Mexico City altitude [65,66]. Oxygen consumption measurements were done in 1 ml of M9 buffer and normalized to the total protein, which was determined as above described in worms recovered from the oxygen chamber at the end of the measurement. To evaluate the mitochondrial contribution to the oxygen consumption rates of the entire animals, we added 1 mM sodium azide for 5 min after starting the measurement.

Analysis of total fatty acid profiles
Total fatty acids were extracted from nematodes essentially following a previous method [68] with heptadecanoic acid (internal standard) and chloroform-methanol (2:1, v/v) [69] having 0.002% n-butyl-hydroxy-toluene as an antioxidant to avoid further oxidation [70]. The generation of fatty-acid methyl esters (FAMEs) was done following an earlier method [29] by dissolving the obtained lipid fraction with a mixture of 0.2 mL toluene, 1.76 mL methanol and 0.04 mL H 2 SO 4 before incubating the samples at 95°C for 1 h. The concentration and composition of FAMEs were evaluated as described previously [71] using a Shimadzu GC-8a gas chromatodph. Helium gas was used as the carrier of the sample and the detection was performed using a H 2 flame. The chromatograph injector was set at 250°C and the initial temperature of the column ranged from 150 to 220°C (final). The peaks of each fatty acid were quantified by comparison with the concentration of the internal standard.