Simple Enzymatic Assay Research Articles

Assay Replicability in β-Glucosidase Enzyme Kinetics Across Laboratories

Replicability is the foundation of research in any scientific discipline. Despite this fact, few studies address experimental variability within and across multiple institutions that operate under the same protocol. While consistency is usually well documented within the same lab, multi-institutional experiments may introduce new variables and, therefore, variability that may lead to inconsistent results. This study seeks to explore intra- and interinstitutional variability among enzyme catalytic efficiency values (KM and Kcat/KM) for the wild type of β-Glucosidase derived from Paenibacillus polymyxa. A standardized protocol for the assay was provided to all institutions that participated in the study. The analysis was conducted using data from 13 laboratories across the United States. The information was collected through the Design2Data CURE database. A total of 132 independent assays of β-Glucosidase were analyzed. Statistical analysis of Kcat/KM , KM, and T50 was conducted using SPSS, and Whisker Plots with a 90% confidence interval were generated for all parameters. Acceptance intervals for the parameters assayed of β-Glucosidase were determined; these could be used in all future experiments in the network as a reference by current and incoming laboratory members that incorporate into the network yearly. Ultimately, we aim to identify possible errors and misunderstandings in the protocol we use in the laboratories of the D2D network to improve assay replicability across institutions. High variability may be a concern when data is interpreted across institutions. This specific assay is used as a control for other, more complex assays in the CURE network and, therefore, the variability found can potentially lead to incorrect interpretation of results. It also highlights the high variability of a simple enzymatic assay when it is replicated in laboratories with different personnel and equipment, regardless of using the same standardized protocol.

The progress of clinical research on the detection of 1,5-anhydroglucitol in diabetes and its complications.

1,5-Anhydroglucitol (1,5-AG) is sensitive to short-term glucose fluctuations and postprandial hyperglycemia, which has great potential in the clinical application of diabetes as a nontraditional blood glucose monitoring indicator. A large number of studies have found that 1,5-AG can be used to screen for diabetes, manage diabetes, and predict the perils of diabetes complications (diabetic nephropathy, diabetic cardiovascular disease, diabetic retinopathy, diabetic pregnancy complications, diabetic peripheral neuropathy, etc.). Additionally, 1,5-AG and β cells are also associated with each other. As a noninvasive blood glucose monitoring indicator, salivary 1,5-AG has much more benefit for clinical application; however, it cannot be ignored that its detection methods are not perfect. Thus, a considerable stack of research is still needed to establish an accurate and simple enzyme assay for the detection of salivary 1,5-AG. More clinical studies will also be required in the future to confirm the normal reference range of 1,5-AG and its role in diabetes complications to further enhance the blood glucose monitoring system for diabetes.

Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis.

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs to produce aminoacyl-tRNAs for protein synthesis. Bacterial aaRSs have distinctive features, play an essential role in channeling amino acids into biomolecular assembly, and are vulnerable to inhibition by small molecules. The aaRSs continue to be targets for potential antibacterial drug development. The first step of aaRS reaction is the activation of amino acid by hydrolyzing ATP to form an acyladenylate intermediate with the concomitant release of pyrophosphate. None-radioactive assays usually measure the rate of ATP consumption or phosphate generation, offering advantages in high-throughput drug screening. These simple aaRS enzyme assays can be adapted to study the mode of inhibition of natural or synthetic aaRS inhibitors. Taking phenylalanyl-tRNA synthetase (PheRS) of Mycobacterium tuberculosis (Mtb) as an example, we describe a process for identification and characterization of Mtb PheRS inhibitor.

Enzymatic measurement of short-chain fatty acids and application in periodontal disease diagnosis.

Periodontal disease is a chronic inflammatory condition caused by periodontal pathogens in the gingival sulcus. Short-chain fatty acids (SCFAs) produced by causal bacteria are closely related to the onset and progression of periodontal disease and have been reported to proliferate in the periodontal sulcus of patients experiencing this pathology. In such patients, propionic acid (C3), butyric acid (C4), isobutyric acid (IC4), valeric acid (C5), isovaleric acid (IC5), and caproic acid (C6), henceforth referred to as [C3-C6], has been reported to have a detrimental effect, while acetic acid (C2) exhibits no detrimental effect. In this study, we established an inexpensive and simple enzymatic assay that can fractionate and measure these acids. The possibility of applying this technique to determine the severity of periodontal disease by adapting it to specimens collected from humans has been explored. We established an enzyme system using acetate kinase and butyrate kinase capable of measuring SCFAs in two fractions, C2 and [C3-C6]. The gingival crevicular fluid (GCF) and saliva of 10 healthy participants and 10 participants with mild and severe periodontal disease were measured using the established enzymatic method and conventional gas chromatography-mass spectrometry (GC-MS). The quantification of C2 and [C3-C6] in human GCF and saliva was well correlated when using the GC-MS method. Furthermore, both C2 and [C3-C6] in the GCF increased with disease severity. However, while no significant difference was observed between healthy participants and periodontal patients when using saliva, [C3-C6] significantly differed between mild and severe periodontal disease. The enzymatic method was able to measure C2 and [C3-C6] separately as well as using the GC-MS method. Furthermore, the C2 and [C3-C6] fractions of GCF correlated with disease severity, suggesting that this method can be applied clinically. In contrast, the quantification of C2 and [C3-C6] in saliva did not differ significantly between healthy participants and patients with periodontal disease. Future studies should focus on inflammation rather than on tissue destruction.

Carbon Dot/Naphthalimide Based Ratiometric Fluorescence Biosensor for Hyaluronidase Detection.

Bladder cancer is the leading cause of death in patients with genitourinary cancer. An elevated level of hyaluronidase (HAase) was found in bladder cancer, which acts as an important biomarker for the early diagnosis of bladder cancer. Hence, there is a need to develop a simple enzymatic assay for the early recognition of HAase. Herein, we report a simple, sensitive, and ratiometric fluorescence assay for HAase detection under physiological conditions. The fluorescence assay was constructed by the adsorption of cationic carbon dots and positively charged naphthalimide on negatively charged hyaluronic acid and the development of a Förster resonance energy transfer (FRET) mechanism from carbon dots to a naphthalimide fluorophores. The hyaluronidase enzyme cleaves the hyaluronic acid in this assay, and breaking down the FRET mechanism induces ratiometric changes. A detection limit of 0.09 U/mL was achieved, which is less than the HAase level found in normal human body fluids. Moreover, this assay may be used for diagnosing HAase-related diseases.

A simple enzymatic assay for the quantification of C1-specific cellulose oxidation by lytic polysaccharide monooxygenases.

ObjectiveThe development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs).ResultsIn combination with a β-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1- oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation.ConclusionsWe conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

An enzymatic assay for quantification of inositol in human term placental tissue

A modified sensitive, cheap and simple enzymatic assay method is described for the quantitation of inositol (6-carbon polyol) in human placental tissue. Water-soluble and total (water-soluble and lipid-bound) inositol isomers were extracted and quantified using a 96-well adaptation of the Megazyme® assay. This assay specifically recognized myo-inositol (predominant isomer), d-chiro-, epi-, and allo-inositols, but not scyllo-inositol, glucose or fucose. In term placenta, water-soluble and total inositol contents were high [489 (±58) and 635 (±69) μg/g respectively], and reliably quantified with good reproducibility. This modified assay could facilitate placental inositol biology research, particularly pertinent now with interest in myo-inositol supplementation for gestational diabetes (GDM) prevention.

Application of high-throughput screening for evaluating hydrolytic potential of cellulases

Heterogeneous nature of cellulose is defiant to enzymatic hydrolysis and individual methods for activity determination of cellulases are inefficient to define actual potency. Hence, three major enzyme activities (FPase, CMCase, CBase) along with protein concentrations were determined to define biomass hydrolyzing potential of cellulases. In present study, tenfold downscaled (modified) assays based on high-throughput screening (HTS) system were developed to increase the throughput and efficiency of enzyme component assays. HTS is a powerful tool which facilitates discovery of new or improved enzymes with appropriate biochemical properties for commercial biorefineries. Cellulase activity of four commercial cellulase preparations was determined and was found to have no significant difference at the 95% confidence level between the values obtained using standard (IUPAC) method and modified microplate method. This modification allowed us to characterize cellulases based on the relative ratios of their individual components and avoids ambiguity concerning potency based on the conventional FPase assay. Also, this work puts forth a simple, rapid, and complete cellulolytic enzyme assay method.

Wax-Printed Fluidic Time Delays for Automating Multi-Step Assays in Paper-Based Microfluidic Devices (MicroPADs)

Microfluidic paper-based analytical devices (microPADs) have emerged as a promising platform for point-of-care diagnostic devices. While the inherent wicking properties of microPADs allow for fluid flow without supporting equipment, this also presents a major challenge in achieving robust fluid control, which becomes especially important when performing complex multi-step assays. Herein, we describe an ideal method of fluid control mediated by wax-printed fluidic time delays. This method relies on a simple fabrication technique, does not utilize chemicals/reagents that could affect downstream assays, is readily scalable, and has a wide temporal range of tunable fluid control. The delays are wax printed on both the top and bottom of pre-fabricated microPAD channels, without subsequent heating, to create hemi-/fully-enclosed channels. With these wax printed delays, we were able to tune the time it took aqueous solutions to wick across a 25 mm-long channel between 3.6 min and 13.4 min. We then employed these fluid delays in the sequential delivery of four dyes to a test zone. Additionally, we demonstrated the automation of two simple enzymatic assays with this fluid control modality. This method of fluid control may allow future researchers to automate more complex assays, thereby further advancing microPADs toward real-world applications.

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

Applicability of API ZYM to capture seasonal and spatial variabilities in lake and river sediments

ABSTRACTWaters draining into a lake carry with them much of the suspended sediment that is transported by rivers and streams from the local drainage basin. The organic matter processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and evaluate some of these variabilities in the sediments, we sampled six sites: three from the St. Clair River and three from Lake St. Clair in spring, summer, fall, and winter of 2016. At all sites and dates, we investigated the spatial and temporal variations in 19 extracellular enzyme activities using API ZYM. Our results indicated that a broad range of enzymes were found to be active in the sediments. Phosphatases, lipases, and esterases were synthesized most intensively by the sediment microbial communities. No consistent difference was found between the lake and sediment samples. Differences were more obvious between sites and seasons. Sites with the highest metabolic (enzyme) diversity reflected the capacity of the sediment microbial communities to breakdown a broader range of substrates and may be linked to differences in river and lake water quality. The seasonal variability of the enzymes activities was governed by the variations of environmental factors caused by anthropogenic and terrestrial inputs, and provides information for a better understanding of the dynamics of sediment organic matter of the river and lake ecosystems. The experimental results suggest that API ZYM is a simple and rapid enzyme assay procedure to evaluate natural processes in ecosystems and their changes.

Graying the self-assembly of gold nanoparticles for improved enzyme activity assays

Despite the simplicity and usability of gold nanoparticle (AuNP)-based colorimetry in a large panel of bioassays, this technique still suffers from a poor detection limit. To overcome this issue, we report an improved AuNP-based colorimetric assay in combination with simple centrifugation and silver enhancement. As a proof-of-concept, two types of enzymes (i.e., protein phosphatase and protease) were constructed based on the self-assembly of thiol-stabilized AuNPs in the presence of peptides and metal ions. When the AuNP solutions were subjected to a short cycle of centrifugation and silver enhancement, the signal-to-background ratio (SBR) was distinctly improved by a factor of 5–10 in both enzyme activity assays, as compared to that of AuNP-based colorimetry. The detection limit achieved by using the silver enhancement was determined to be improved by a factor of 1.0–3.4. Furthermore, grayscale images of the silver enhancement allowed for a rapid and simple enzyme assay that did not require measuring the absorbance. Due to its ability to improve the detection senstivity in a facile way, we anticipate that this approach will be suitable for use in many AuNP colorimetric assays.

A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes.

The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the E. coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured KM values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a kcat of 7.4–9.0 s-1 with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed KM values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a kcat of 2100–2600 s-1 for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.

Quantitative Analytical Method for the Determination of Biotinidase Activity in Dried Blood Spot Samples.

Biotinidase activity assay is included in most newborn screening protocols, and the positive results are confirmed by quantitative enzyme activity measurements. In our study, we describe a new quantitative analytical method for the determination of biotinidase activity using the blood sample deposited onto filter paper as the assay medium, by predepositing N-biotinyl-p-aminobenzoic acid onto the standard sample collection paper. The analysis of the assay mixture requires a simple extraction step from a dried blood spot followed by the quantification of product by LC-MS. The method provides a simple and reliable enzyme assay method that enables the rapid diagnosis of biotinidase deficiency (BD). Out of the measured 36 samples, 13 were healthy with lower enzyme activities, 16 were patients with partial BD, and 7 were patients with profound BD with residual activity below 10%. Expression of enzyme activity in percentage of mean activity of negative controls allows comparison of the different techniques. The obtained results are in good agreement with activity data determined from both dried blood spots and serum samples, giving an informative diagnostic value.

Many disease-associated variants of hTERT retain high telomerase enzymatic activity

Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays.

Evaluation of etravirine resistance in clinical samples by a simple phenotypic Test

Drug resistance testing is an important tool in the management of HIV-1 infection. As access to genotypic resistance assays is limited in low- and middle-income settings, alternatives are warranted. The aim of the study was to adapt a phenotypic drug susceptibility assay, ExaVir Drug, for detection of resistance to the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) etravirine (ETR). Five NNRTI resistant mutant forms of RT were produced (L100I, K103N, L100I/K103N, Y181C, V179D) in order to validate the assay for ETR. Furthermore, HIV-1 RT was purified from plasma samples (n = 28) obtained from treatment naïve and experienced HIV-1 infected patients, and ETR drug susceptibility (IC(50)) was estimated. The direct sequencing of the pol gene was performed. The recombinant RT mutants had the expected changes in drug sensitivity patterns. The RTs isolated from plasma of therapy naïve individuals showed low IC(50) for ETR. In the plasma virus from treatment experienced patients with Y181C, A98G, V108I, and/or K101E mutations in the pol gene, higher IC(50) values were found in line with reduced susceptibility data for ETR. This study demonstrates that ExaVir® Drug, a simple enzymatic phenotypic assay, can be used for detection of ETR resistance, including cross-resistance to other NNRTIs, in clinical samples. A further evaluation is needed to define clinical cut-offs; however the assay is an alternative to more costly HIV drug resistance tests, especially in low-income countries.

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20nM to 50μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy.

Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-D: -glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC(50)) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease.

Mitochondrial malic enzyme (ME2) in pancreatic islets of the human, rat and mouse and clonal insulinoma cells: Simple enzyme assay for mitochondrial malic enzyme 2

Despite interest in malic enzyme(ME)s in insulin cells, mitochondrial malic enzyme (ME2) has only been studied with estimates of mRNA or with mRNA knockdown. Because an mRNA’s level does not necessarily reflect the level of its cognate enzyme, we designed a simple spectrophotometric enzyme assay to measure ME2 activity of insulin cells by utilizing the distinct kinetic properties of ME2. Mitochondrial ME2 uses either NAD or NADP as a cofactor, has a high K m for malate and is allosterically activated by fumarate and inhibited by ATP. Cytosolic ME (ME1) and the other mitochondrial ME (ME3) use only NADP as a cofactor and have lower K ms for malate. The assay easily showed for the first time that substantial ME2 activity is present in pancreatic islets of humans, rats and mice and INS-1 832/13 cells. ME2’s presence was confirmed with immunoblotting. There was no evidence that ME3 is present in these tissues.