Nothing
Intrinsic Hepatic Clearance (Cl~int~)"
In invitroTKstats: In Vitro Toxicokinetic Data Processing and Analysis Pipeline
```{css,code = readLines(params$css),hide=TRUE,echo=FALSE}
```r
knitr::opts_chunk$set(
collapse = TRUE,
comment = "#>",
fig.align = 'center'
)
Introduction
This vignette guides users on how to estimate intrinsic hepatic clearance (Cl~int~) from mass spectrometry data. Intrinsic hepatic clearance is a chemical specific parameter that describes a liver's ability to metabolize an unbound compound. It is characterized by the disappearance of compound when incubated with primary hepatocytes (@breen2021high).
The mass spectrometry data should be collected from an assay that uses cryopreserved hepatocyte suspension (@shibata2002prediction) as seen in Figure 1.
knitr::include_graphics("img/clint_diagram.png")
Suggested packages for use with this vignette
# Primary package
library(invitroTKstats)
# Data manipulation package
library(dplyr)
# Table formatting package
library(flextable)
Load Data
First, we load in the example dataset from invitroTKstats.
# Load example clint data
data("Clint-example")
Many datasets are loaded in: clint_L0, clint_L1,clint_L2, clint_L3, and clint_L4. These datasets are Cl~int~ data at Level 0, 1, 2, 3, and 4 respectively. Additional datasets associated with Level 4 processing are also loaded in: clint_L2_heldout and clint_PREJAGS. These will be described later in the "Level 4 processing" section.
Lastly, a clint_cheminfo dataset is loaded in that contains chemical information necessary for identification mapping; it is used to create Level 0 data. For the purpose of this vignette, we'll start with clint_L0, the Level 0 data, to demonstrate the complete pipelining process.
clint_L0 is the output from the merge_level0 function which compiles raw lab data from specified Excel files into a singular data frame. The data frame contains exactly one row per sample with information obtained from the mass spectrometer. For more details on curating raw lab data to a singular Level 0 data frame, see the "Data Guide Creation and Level-0 Data Compilation" vignette.
The following table displays the first three rows of clint_L0, our Level 0 data.
head(clint_L0, n = 3) %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
)
)
) %>%
set_caption(caption = "Table 1: Level 0 data",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
Level 1 processing
format_clint is the Level 1 function used to create a standardized data frame. This level of processing is necessary because naming conventions or formatting can differ across data sets.
If the Level 0 data already contains the required column, then the existing column name can be specified. For example, clint_L0 already contains a column specifying the sample name called "Sample". However, the default column name for sample name is "Lab.Sample.Name". Therefore, we specify the correct column with sample.col = "Sample". In general, to specify an already existing column that differs from the default, the user must use the parameter with the .col suffix.
If the Level 0 data does not already contain the required column, then the entire column can be populated with a single value. For example, clint_L0 does not contain a column specifying biological replicates. Therefore, we populate the required column with biological.replicates = 1. In general, to specify a single value for an entire column, the user must use the parameter without the .col suffix.
Users should be mindful if they choose to specify a single value for all of their samples; they should verify this action is one they wish to take.
Some columns must be present in the Level 0 data while others can be filled with a single value. At minimum, the following columns must be present in the Level 0 data and specification with a single entry is not permitted: sample.col, compound.col, dtxsid.col, lab.compound.col, type.col, istd.col, and area.col.
If there is no additional note.col in the Level 0 data, users should use note.col = NULL to fill the column with "Note".
The rest of the following columns may either be specified from the Level 0 data or filled with a single value: date.col or date, density.col or density, cal.col or cal, dilution.col or dilution, time.col or time, istd.name.col or istd.name, istd.conc.col or istd.conc, test.conc.col or test.conc, test.nominal.conc.col or test.nominal.conc, biological.replicates.col or biological.replicates, analysis.method.col or analysis.method, analysis.instrument.col or analysis.instrument, analysis.parameters.col or analysis.parameters, level0.file.col or level0.file, and level0.sheet.col or level0.sheet.
# Create table of required parameters for Level 1
req_cols = data.frame(matrix(nrow = 31, ncol = 5))
vars <- c("Argument", "Default", "Required in L0?", "Corresp. single-entry Argument", "Descr.")
colnames(req_cols) <- vars
# Argument names
arguments <- c("FILENAME", "data.in", "sample.col", "date.col", "compound.col",
"dtxsid.col", "lab.compound.col", "type.col", "density.col",
"cal.col", "dilution.col", "time.col", "istd.col", "istd.name.col",
"istd.conc.col", "test.conc.col", "test.nominal.conc.col",
"area.col", "biological.replicates.col", "technical.replicates.col",
"analysis.method.col", "analysis.instrument.col",
"analysis.parameters.col", "note.col", "level0.file.col",
"level0.sheet.col", "output.res", "save.bad.types", "sig.figs", "INPUT.DIR",
"OUTPUT.DIR")
req_cols[,"Argument"] <- arguments
# Default arguments
defaults <- c("MYDATA", NA, "Lab.Sample.Name", "Date", "Compound.Name",
"DTXSID", "Lab.Compound.Name", "Sample.Type", "Hep.Density",
"Cal", "Dilution.Factor", "Time", "ISTD.Area", "ISTD.Name", "ISTD.Conc",
"Test.Compound.Conc", "Test.Target.Conc", "Area",
"Biological.Replicates", "Technical.Replicates", "Analysis.Method",
"Analysis.Instrument", "Analysis.Parameters", "Note",
"Level0.File", "Level0.Sheet", "FALSE", "FALSE", 5, NA, NA)
req_cols[,"Default"] <- defaults
# Argument required in L0?
req_cols <- req_cols %>%
mutate("Required in L0?" = case_when(
Argument %in% c("sample.col","compound.col", "dtxsid.col", "lab.compound.col", "type.col", "istd.col", "area.col") ~ "Y",
Argument %in% c("FILENAME", "data.in", "output.res", "save.bad.types", "sig.figs", "INPUT.DIR", "OUTPUT.DIR") ~ "N/A",
.default = "N"
))
# Corresponding single-entry Argument
req_cols <- req_cols %>%
mutate("Corresp. single-entry Argument" = ifelse(.data[[vars[[3]]]] == "N" & .data[[vars[[1]]]] != "note.col", gsub(".col","",Argument),
NA))
# Brief description
description <- c("Output and input filename",
"Level 0 data frame",
"Lab sample name",
"Lab measurement date",
"Formal test compound name",
"EPA's DSSTox Structure ID",
"Lab test compound name (abbr.)",
"Sample type (Blank/Cvst/Inactive/CC)",
"Hepatocyte density in the in vitro incubation",
"MS calibration",
"Number of times sample was diluted",
"Time since chemical was introduced",
"Internal standard peak area",
"Internal standard name",
"Internal standard concentration",
"Standard test chemical concentration",
"Initial chemical concentration",
"Target analyte peak area",
"Replicates with the same analyte",
"Repeated measurements from one sample",
"Analytical chemistry analysis method",
"Analytical chemistry analysis instrument",
"Analytical chemistry analysis parameters",
"Additional notes",
"Raw data filename",
"Raw data sheet name",
"Export results (TSV)?",
"Export bad data (TSV)?",
"Number of significant figures",
"Input directory of Level 0 file",
"Export directory to save Level 1 files"
)
req_cols[,"Descr."] <- description
req_cols %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
height = 200
)
)
) %>%
set_caption(caption = "Table 2: Level 1 'format_clint' function arguments",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
A TSV file containing the level-1 data can be exported to the user's per-session temporary directory. This temporary directory is a per-session directory whose path can be found with the following code: tempdir(). For more details, see
[https://www.collinberke.com/til/posts/2023-10-24-temp-directories/].
To avoid exporting to this temporary directory, an OUTPUT.DIR must be specified. We have omitted this export entirely with output.res = FALSE (the default). The option to omit exporting a TSV file is also available at levels 2 and 3 and will be used from this point forward.
clint_L1_curated <- format_clint(FILENAME = "Clint_vignette",
data.in = clint_L0,
# columns present in L0 data
sample.col = "Sample",
compound.col = "Compound",
lab.compound.col = "Lab.Compound.ID",
type.col = "Type",
istd.col = "ISTD.Peak.Area",
area.col = "Peak.Area",
note.col = "Sample Text",
test.conc.col = "Compound.Conc",
analysis.parameters.col = "Analysis.Params",
# columns not present in L0 data
density = 0.5,
test.nominal.conc = 1,
analysis.instrument = "Unknown",
analysis.method = "LCMS",
istd.conc = 0.01,
cal = 1,
biological.replicates = 1,
# don't export output TSV file
output.res = FALSE
)
We receive a warning message that some of our samples have been removed due to "inappropriate sample types". The removed samples include data that did not have one of the following sample types
- Blank with no chemical added (Blank)
- Hepatocyte incubation concentration (Cvst)
- Inactivated Hepatocytes (Inactive)
- Calibration Curve (CC)
The following table displays some of the samples that were removed. These samples are annotated with a "QC" sample type indicating "Quality Control" and should therefore not be included in our analysis.
clint_L0 %>%
filter(Type == "QC") %>%
head(n = 5) %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
height = 200
)
)
) %>%
set_caption(caption = "Table 3: Subset of removed samples",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
Users can verify that these samples are excluded by filtering their Level 1 data frame and ensuring no matches are found. One approach is given below.
# All the samples of an inappropriate sample type
excluded <- clint_L0 %>%
filter(!Type %in% c("Blank", "Cvst", "Inactive", "CC"))
# Exclude based on Sample and DTXSID
X <- c(excluded$Sample)
names(X) <- paste(excluded$Sample, excluded$DTXSID, sep = "+")
Y <- c(excluded$DTXSID)
# Find samples in L1 data frame with matching sample name (X) and DTXSID (Y)
matches <- as.data.frame(t(mapply(function(X,Y)
{subset(clint_L1_curated, Lab.Sample.Name == X & DTXSID == Y)},
X, Y, USE.NAMES = T)))
matches
# Check that no matches were returned for each sample in `excluded`
check <- rep(1,nrow(matches))
names(check) <- rownames(matches)
for (name in rownames(matches)) {
# If no matches were found, each element of check should evaluate to 0
check[name] <- sum(sapply(c(1:ncol(matches)),
function(X) {length(matches[1,X][[1]])}))
}
# Verify that each sample in `excluded` had no matches
check
# If no matches, should evaluate to 0
sum(check)
To export the removed samples types as a TSV, the user can set the parameter save.bad.types = TRUE. For this example, we will not save the discarded sample types.
The following table displays the first three rows of clint_L1_curated, our Level 1 data produced from format_clint. In addition to the columns specified by the user, there is an additional column called Response. This column is the test compound concentration and is calculated as $\textrm{Response} = \frac{\textrm{Analyte Area}}{\textrm{ISTD Area}}*\textrm{ISTD Conc}$ where $\textrm{Analyte Area}$ is defined by the Area column, $\textrm{ISTD Area}$ is defined by the ISTD.Area column, and $\textrm{ISTD Conc}$ is defined by the ISTD.Conc column.
clint_L1_curated %>%
head(n = 3) %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
)
)
) %>%
set_caption(caption = "Table 4: Level 1 data",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
Level 2 processing
sample_verification is the Level 2 function used to add a verification column. The verification column indicates whether a sample should be included in the point estimation (Level 3) and credible interval (Level 4) processing. This column allows users to keep all samples in their data but only utilize the reliable samples for Cl~int~ estimation. All of the data in Level 2 is identical to the data in Level 1 with the exception of the additional Verified column.
To determine whether a sample should be included, the user should consult the wet-lab scientists from where their data originates or a chemist who may be able to provide reliable rationale for samples that should not be verified. This level of processing allows the user to receive feedback from the wet-lab scientists, exclude erroneous or unreliable samples, and produce new Cl~int~ estimates. Thus, there is always an open channel of communication between the user and the wet-lab scientists or chemists.
We will use the already processed Level 2 data frame, clint_L2, to regenerate our exclusion data. In general, the user would not have access to the exclusion information a priori.
The exclusion data frame must include the following columns: Variables, Values, and Message. The Variables column contains the variable names used to filter the excluded rows. Here, we are using Lab.Sample.Name and DTXSID to identify the excluded rows separated by a "|". The Values column contains the values of the variables, as a character, also separated by a "|". The Message column contains the reason for exclusion. Here, we are using the reasons listed in the Verified column in clint_L2. The user should refrain from using "|" in any of their descriptions to avoid conflicts with the sample_verification function.
# Use verification data from loaded in `clint_L2` data frame
exclusion <- clint_L2 %>%
filter(Verified %in% c("Wrong Mix", "Unknown Conc.")) %>%
mutate("Variables" = "Lab.Sample.Name|DTXSID") %>%
mutate("Values" = paste(Lab.Sample.Name, DTXSID, sep = "|")) %>%
mutate("Message" = Verified) %>%
select(Variables, Values, Message)
exclusion %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
height = 200
)
)
) %>%
set_caption(caption = "Table 5: Exclusion data frame",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
clint_L2_curated <- sample_verification(FILENAME = "Clint_vignette",
data.in = clint_L1_curated,
assay = "Clint",
exclusion.info = exclusion,
# don't export output TSV file
output.res = FALSE)
Our Level 2 data now contains a Verified column. If the sample should be included, the column contains a "Y" for yes. If the sample should be excluded, the column contains the reason for exclusion.
The following table displays some rows of the Level 2 data, each with a different value in the Verification column. Note, the reasons for exclusion are not limited to "Unknown Conc." or "Wrong Mix", they were merely provided as an example.
df1 <- head(clint_L2_curated, n = 1)
df2 <- head(filter(clint_L2_curated, Verified == "Wrong Mix"), n = 1)
df3 <- head(filter(clint_L2_curated, Verified == "Unknown Conc."), n = 1)
temp <- rbind(df1, df2)
temp <- rbind(temp, df3)
temp %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
)
)
) %>%
set_caption(caption = "Table 6: Level 2 data",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
Level 3 processing
calc_clint_point is the Level 3 function used to calculate the Cl~int~ point estimate for each test compound using a Frequentist framework.
Mathematically, Cl~int~ is the slope of the line representing log concentrations over time. With some assumptions on the form of the line and on the distribution of the error terms, we can estimate Cl~int~ using maximum likelihood estimation.
First, we assume the test compound concentration at time $t$ is an exponential decay model $$C(t) = \textrm{cal}C_0e^{-mt}$$ where $\textrm{cal}$ is the MS calibration, $C_0$ is the test compound concentration at time 0, $m$ is the rate constant, and $t$ is the incubation time. Notice that if a natural log is applied, $$\textrm{ln}(C(t)) = -mt + \textrm{ln}(\textrm{cal}*C_0)$$ $m$ is the slope of the line representing log concentrations over time. Thus, the rate constant $m$ is our Cl~int~ value.
Next, we also assume the random noise is normally distributed $\varepsilon_i \sim \mathcal{N}(0,\sigma^2)$ where $\sigma^2$ is unknown.
Lastly, the test compound concentration, our dependent variable, is qualitatively defined by our Response column, the ratio of the analyte area to the ISTD area.
With this information, we can rewrite our dependent variable as $$y_i \sim \mathcal{N}(C(t),\sigma^2)$$ After some simplification, our log-likelihood is defined as $$l(\theta) = N*log\left(\frac{1}{\sqrt{2\pi}\sigma}\right)-\frac{1}{2}\sum_{i=1}^N\frac{(y_i-C(\theta, t))^2}{\sigma^2}$$ where $\theta = (\textrm{cal}, m)$ and $N$ is the number of Cvst and Blank samples.
Using the mle estimator in the stats4 package, we then estimate the parameters that maximize the log-likelihood for each test compound.
clint_L3_curated <- calc_clint_point(FILENAME = "Clint_vignette",
data.in = clint_L2_curated,
# don't export output TSV file
output.res = FALSE)
Our Level 3 data contains a Clint estimate, Clint.pValue, AIC of the exponential decay fit, and AIC.Null of the exponential decay assuming a constant rate of decay.
Some experiments also include the assay performed at 1 $\mu M$ and 10 $\mu M$ to check for the saturation of metabolizing enzymes. The test compound concentration at time $t$ with a saturation probability is assumed to follow an exponential decay model with a slight modification $$C_{sat}(t) = \textrm{cal}C_0e^{-m\textrm{sat}t}$$ where $\textrm{cal}$ is the MS calibration, $C_0$ is the test compound concentration at time 0, $m$ is the rate constant, $t$ is the incubation time, and $\textrm{sat}$ is the probability of saturation, or the probability of observing a lower clearance rate at a higher concentration.
If these samples are included, Clint.1 is the clearance rate at 1 $\mu M$, Clint.10 is the clearance rate at 10 $\mu M$, AIC.Sat is the AIC of the exponential decay with a saturation probability, and Sat.pValue is the corresponding p-value.
Because our example data did not include these samples, Clint.1, Clint.10, AIC.Sat, and Sat.pValue are empty.
clint_L3_curated %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
)
)
) %>%
set_caption(caption = "Table 7: Level 3 data",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
Level 4 processing
calc_clint is the Level 4 function used to calculate Cl~int~ point estimates and credible intervals using a Bayesian framework. Markov chain Monte Carlo (MCMC) simulations are used to randomly sample from the posterior distribution with a uniform prior.
To run Level 4, one needs to have JAGS installed on their machine. To determine the correct path, the user may use the runjags::findjags() or may need to specify the JAGS installation location for the JAGS.PATH argument.
:::{.noticebox}
JAGS is a software used for conducting Bayesian hierarchical modeling using Markov Chain Monte Carlo (MCMC) simulation. invitroTKstats contains the JAGS models for each of the applicable assays and utilizes the JAGS model under the hood to run the MCMC simulations via the runjags dependency to obtain the level-4 Bayesian estimates.
- Download and install the "Just Another Gibbs Sampler" (JAGS) software, which is freely available here.
- Once JAGS is installed one will need to identify the installation location, which is likely to be in a system location (e.g. "Program Files"). Users may need this location to specify the
JAGS.PATH argument within the level-4 function.
- Finally, if not already installed, the
rjags, runjags, and coda R packages need to be installed via the R console using the install.packages function.
:::
We pass clint_L2_curated, the Level 2 data frame, and not clint_L3_curated, the Level 3 data frame, into calc_clint. This is because Level 3 and Level 4 processing are not sequential; they are methods that calculate different statistical quantities. The following code chunk takes a while to run; previous runtimes are around 7 minutes.
clint_L4_curated <- calc_clint(FILENAME = "Clint_vignette",
data.in = clint_L2_curated,
JAGS.PATH = runjags::findjags()
)
clint_L4_curated <- clint_L4
The Cl~int~ intervals are returned to the user's R session, in an exported TSV file, and in an exported RData file. There is no parameter to prevent the TSV or RData files from being exported because of the potential for the simulations to crash. If there are no crashes, then the exported TSV file is identical to the user's R session and the exported RData file. clint_L4 is an example exported RData file.
Additionally, intermediate files are saved to the user's current working directory if TEMP.DIR = NULL. These include a Level 2 heldout set, clint_L2_heldout, containing unverified samples and a Level 4 PREJAGS list, clint_PREJAGS, containing arguments provided to JAGS. Because the PREJAGS list is overwritten with each compound, clint_PREJAGS only contains information relevant to the last tested compound, TFMFPA in this case.
Our Level 4 data contains a Cl~int~ credible interval and the corresponding p-value for each chemical.
clint_L4_curated %>%
flextable() %>%
bg(bg = "#DDDDDD", part = "header") %>%
autofit() %>%
set_table_properties(
opts_html = list(
scroll = list(
)
)
) %>%
set_caption(caption = "Table 8: Level 4 data",
align_with_table = FALSE) %>%
fontsize(size = 10, part = "all") %>%
theme_vanilla()
Best Practices: Food for thought
Generally, data processing pipelines should include minimal to no manual coding. It is best to keep clean code that is easily reproducible and transferable. The user should aim to have all the required data and meta-data files properly formatted to avoid further modifications throughout the pipeline.
Appendix
L4 Bayesian Modeling Information
In this section, we provide the equations for the Bayesian model (i.e., priors and likelihoods) used to estimate the intrinsic hepatic clearance ($Cl_{int}$) and the uncertainty about that estimate. The following sub-sections are organized such that:
- Each section contains the relevant hyper-parameters and their priors followed by the variable likelihoods
- Sections are ordered from the most fundamental variables/parameters (i.e. those used in later equations) to the likelihood for the observations used to estimate the intrinsic hepatic clearance ($Cl_{int}$)
Some of the indices are reused between sections (e.g. $i$, $w^*_i$, etc.). However, it should be noted that these are not meant to be understood across sub-sections; rather, only understood within the context of the section they are in.
NOTE: Readers unfamiliar with JAGS should be aware that JAGS software uses precision ($\tau$) rather than variance ($\sigma^2$) (i.e., $\tau = \frac{1}{\sigma^2}$).
Measurement Model
Each chemical may have more than one day of experimentation, and thus multiple calibrations. Suppose for the chemical of interest there are a total of $n_{cal}$ calibrations (Num.cal). For a particular calibration $w \in (1,\ldots,n_{cal})$ we assume the following priors for our Bayesian model:
Prior for log-scale constant analytic standard deviation (log.const.analytic.sd):
$$ log(\sigma_a)w \sim \textrm{Unif} \left( a = -6,b = 1 \right) ; \space \sigma{a,w} = 10^{log(\sigma_a)_w} $$
where $a$ and $b$ are the minimum and maximum, respectively, and $\sigma_{a,w}$ is the converted parameter used in later equations (const.analytic.sd).
Prior for the log-scale heteroscedastic analytic slope (log.hetero.analytic.slope):
$$ log(m_h)w \sim \textrm{Unif} \left( a = -6,b = 1 \right) ; \space m{h,w} = 10^{log(m_h)_w} $$
where $a$ and $b$ are the minimum and maximum, respectively, and $m_{h,w}$ is the converted parameter used in later equations (hetero.analytic.slope).
Prior for the threshold concentration (C.thresh):
$$ C_{thresh,w} \sim \textrm{Unif} \left( a = 0,b = \frac{conc_{target,w}}{10} \right)$$
where $a$ and $b$ are the minimum and maximum, respectively, and $conc_{target,w}$ is the expected initial concentration (Test.Nominal.Conc) for calibration index $w$.
Prior for the log-scale calibration (log.calibration):
$$ log(cal)_w \sim \textrm{N} \left( \mu = 0,\tau = 0.01 \right) ; \space cal_w = 10^{log(cal)_w}$$
where $\mu$ and $\tau$ are the mean and precision, respectively, and $cal_w$ is the converted parameter used in later equations (calibration).
Prior for the background (background):
$$ \gamma_w \sim \textrm{Exp} \left( \lambda = 100 \right)$$
where $\lambda$ is the rate parameter.
Estimation of Blanks
Let us assume the total number of prediction and precision for the blank observations is based on the total number of calibrations in the set (i.e. $n_{cal}$, Num.cal). Thus, for each calibration $j \in w$ we obtain the following:
Estimation for the predicted values for blank observations (Blank.pred):
$$ x_{blank,j} = \frac{\gamma_{j}}{df_{blank,j}} $$
where $df_{blank,j}$ is the dilution factor for the $j^{th}$ calibration for blank observations (Blank.Dilution.Factor).
Estimation for the precision of blank observations (Blank.prec):
$$ \tau_{blank,j} = \frac{1}{(\sigma_{a,j} + m_{h,j} *x_{blank,j})^2} $$
Likelihood for blank observations (Blank.obs):
Suppose $n_{blank}$ is the total number of blank observations (Num.blank.obs), and $y_{blank,i}$ indicates the $i^{th}$ blank observation, sample type = "Blank" (Blank.obs). For each observation we obtain a posterior MCMC estimate for the blank observations with the following:
$$ y_{blank,i} \sim \textrm{N} \left( \mu = x_{blank,w^i}, \tau = \tau{blank,w^_i} \right) $$
where $w^_i$ is the calibration index (Blank.cal) for the $i^{th}$ observation, such that $w^ = (w^_1,\ldots,w^{n{blank}})$ and $w^_i \in w$ (i.e. $w^i$ indicates one of the $n{cal}$ calibrations).
Estimation of Calibration Curve
Suppose $n_{cc}$ is the total number of calibration curve observations (Num.cc), and $y_{cc,i}$ indicates the $i^{th}$ calibration curve observation, sample type = CC (cc.obs). For each observation we obtain a posterior MCMC estimate for the calibration curve observations with the following:
Calibration curve slope for the $i^{th}$ observation (cc.slope) is assumed to be the calibration estimate corresponding to the calibration index ($w^*_i$):
$$ m_{cc,i} = cal_{w^*_i}$$
where $w^_i$ is the calibration index (cc.obs.cal) for the $i^{th}$ observation, such that $w^ = (w^_1,\ldots,w^{n{cc}})$ and $w^_i \in w$ (i.e. $w^i$ indicates one of the $n{cal}$ calibrations).
Calibration curve intercept for the $i^{th}$ observation (cc.intercept) is assumed to be the background estimate corresponding to the calibration index ($w^*_i$):
$$ \alpha_{cc,i} = \gamma_{w^*_i}$$
Estimation of the predictions for calibration curve observations (cc.pred):
$$ x_{cc,i} = m_{cc,i} * (\frac{C_{cc,i}}{df_{cc,i}} - \frac{C_{thresh,w^*i}}{df{cc,i}}) * \beta_{cc,i} + \frac{\alpha_{cc,i}}{df_{cc,i}} $$
where
- $C_{cc,i}$ is the chemical concentration (
cc.obs.conc) for the $i^{th}$ calibration curve observation
- $df_{cc,i}$ is the dilution factor (
cc.obs.Dilution.Factor) for the $i^{th}$ calibration curve observation
- $\beta_{cc,i}$ is the JAGS step function for the $i^{th}$ calibration curve observation, such that
$$
\beta_{cc,i} =
\begin{cases}
1 & \textrm{if} \space (\frac{C_{cc,i}}{df_{cc,i}} - \frac{C_{thresh,w^*i}}{df{cc,i}}) \ge 0 \
0 & \textrm{otherwise}
\end{cases}
$$
Estimation of the precision for calibration curve observations (cc.prec):
$$ \tau_{cc,i} = \frac{1}{(\sigma_{a,w^i} + m{h,w^i} * x{cc,i})^2} $$
Likelihood for the calibration curve observations (cc.obs):
$$ y_{cc,i} \sim \textrm{N} \left( \mu = x_{cc,i},\tau = \tau_{cc,i} \right)$$
Clearance Model
In some cases, the intrinsic hepatic clearance assay will have two initial concentrations (i.e. lower and higher concentration), typically these are $1$ and $10 \mu M$ but do not necessarily need to be.
Prior for the binary indicator for whether the concentration decreases (decreases):
$$ d_c \sim \textrm{Bern} \left( p = p_{d_c} \right) $$
where $p$ is the probability parameter and $p_{d_c}$ is the user-specified prior probability that a chemical will decrease in the assay (DECREASE.PROB).
Prior for the binary indicator for whether the concentration degrades (degrades):
$$ d_g \sim \textrm{Bern} \left( p = p_{d_g} \right) $$
where $p$ is the probability parameter and $p_{d_g}$ is the user-specified prior probability of instability for a chemical within the assay (i.e. abiotic degradation) (DEGRADE.PROB).
Prior for the slope representing the biological clearance rate (bio.rate) for the lower concentration, indicated by $C_{thresh,1}$ and $conc_{target,1}$:
$$ \delta_{bio} \sim \textrm{Unif} \left( a = 0,b = -log(\frac{C_{thresh,1}}{conc_{target,1}}) \right)$$
where $a$ and $b$ are the minimum and maximum, respectively.
Prior for the slope representing the abiotic clearance rate, when data from the inactivated hepatocytes is available (abio.rate) at the lower concentration, indicated by $C_{thresh,1}$ and $conc_{target,1}$:
$$ \delta_{abio} \sim \textrm{Unif} \left( a = 0,b = -log(\frac{C_{thresh,1}}{conc_{target,1}}) \right) $$
where $a$ and $b$ are the minimum and maximum, respectively.
Prior for the binary indicator for whether there is a reduction in the biological clearance and the chemical shows some metabolic saturation between the lower and higher concentration (saturates):
$$ s_f \sim \textrm{Bern} \left( p = p_{s_f} \right)$$
where $p$ is the probability parameter and $p_{s_f}$ is the user-specified prior probability that a chemical's rate of metabolism will decrease between the lower and higher concentration (SATURATE.PROB).
$p_{s_f} = 0.25$ assumes $25\%$ of the chemical shows some metabolic saturation between the lower and higher concentrations.
Prior for the saturation at the higher concentration (i.e. magnitude of decreased biological clearance) at the higher concentration (saturation):
$$ s_a \sim \textrm{Unif} \left( a = 0,b = 1 \right) $$
Estimation of the actual biological clearance rate (bio.slope) at the lower ($m_{bio,1}$) and higher ($m_{bio,2}$) initial concentrations, respectively:
$$ m_{bio,1} = d_c * \delta_{bio} $$
$$ m_{bio,2} = d_c * \delta_{bio} (1-s_fs_a) $$
such that $m_{bio} = (m_{bio,1},m_{bio,2})$.
The total elimination rate (slope) at the lower ($m_1$) and higher ($m_2$) initial concentrations, respectively:
$$ m_1 = d_c * \delta_{bio} + d_g * \delta_{abio} $$
$$ m_2 = d_g * \delta_{abio} + d_c * \delta_{bio} (1-s_fs_a) $$
such that $m = (m_1,m_2)$.
Estimate of the abiotic clearance rate (abio.slope):
$$ m_{abio} = d_g * \delta_{abio} $$
Estimation of Metabolic Clearance
Suppose $n$ is the total number of observations (Num.obs), and $y_{i}$ indicates the $i^{th}$ observation, sample type = Cvst (obs). For each observation we obtain a posterior MCMC estimate for the calibration curve observations with the following:
Estimate of the metabolic exponential decay for the $i^{th}$ observation (C):
$$ C_i = conc_{target,c^_i}exp(-m_{c^_i}t_i) $$
where
- $c^_i$ represents the initial concentration index (
obs.conc) for the $i^{th}$ observation (i.e, $c^ = (c^_1,\ldots,c^_n)$) and $c^*_i \in (1,2)$ such that $1$ indicates the lower initial concentration and $2$ indicates the higher initial concentration
- $conc_{target,c^_i}$ is the expected initial concentration (
Test.Nominal.Conc) corresponding to the $i^{th}$ observation, given the concentration index $c^_i$
- $m_{c^i}$ is the total elimination rate slope for the initial concentration corresponding to the $i^{th}$ observation and $m{c^_i} \in m$
- $t_i$ is the time (
obs.time) for the $i^{th}$ observation
Estimate of the metabolic clearance predictions (obs.pred):
$$ x_i = cal_{w^i} * (\frac{C_i}{df_i} - \frac{C{thresh,w^i}}{df_i}) * \beta_i + \frac{\gamma{w^*_i}}{df_i} $$
where
- $df_i$ is the dilution factor (
obs.Dilution.Factor) for the $i^{th}$ observation
- $\beta_i$ is the JAGS step function for the $i^{th}$ observation, such that
$$
\beta_i =
\begin{cases}
1 & \textrm{if} \space (\frac{C_i}{df_i} - \frac{C_{thresh,w^*_i}}{df_i}) \ge 0 \
0 & \textrm{otherwise}
\end{cases}
$$
- $w^_i$ represents the calibration index (
obs.cal) for the $i^{th}$ observation $w^ = (w^_1,\ldots,w^{n})$ and $w^_i \in w$ (i.e. $w^_i$ indicates one of the $n{cal}$ calibrations)
Estimates of the metabolic clearance precision (obs.prec):
$$ \tau_i = \frac{1}{(\sigma_{a,w^i} + m{h,w^_i}*x_i)^2} $$
Likelihood for metabolic clearance (obs):
$$ y_i \sim \textrm{N} \left( \mu = x_i,\tau = \tau_i \right)$$
Estimation of Abiotic Clearance
Suppose $n_{abio}$ is the total number of abiotic observations (Num.abio.obs), and $y_{abio,i}$ indicates the $i^{th}$ observation, sample type = Inactive (abio.obs). For each observation we obtain a posterior MCMC estimate for the calibration curve observations with the following:
Estimate of the abiotic exponential decay for the $i^{th}$ observation (abio.C):
$$ C_{abio,i} = conc_{target,w^i} * exp(-m{abio}t_{abio,i}) $$
- $w^_i$ represents the calibration index (
abio.obs.cal) for the $i^{th}$ non-biological observation $w^ = (w^_1,\ldots,w^{n_abio})$ and $w^_i \in w$ (i.e. $w^_i$ indicates one of the $n{cal}$ calibrations)
- $conc_{target,w^_i}$ is the expected initial concentration (
Test.Nominal.Conc) corresponding to the $i^{th}$ observation, given the calibration index $w^_i$
- $t_{abio,i}$ is the time for the $i^{th}$ abiotic observation (
abio.obs.time)
Estimates for the abiotic clearance predictions (abio.obs.pred):
$$ x_{abio,i} = cal_{w^i} * (\frac{C{abio,i}}{df_{abio,i}} - \frac{C_{thresh,w^i}}{df{abio,i}}) * \beta_{abio,i} + \frac{\gamma_{w^*i}}{df{abio,i}} $$
where
- $df_{abio,i}$ is the dilution factor (
abio.obs.Dilution.Factor) for the $i^{th}$ abiotic observation
- $\beta_{abio,i}$ is the JAGS step function for the $i^{th}$ abiotic observation, such that
$$
\beta_{abio,i} =
\begin{cases}
1 & \textrm{if} \space (\frac{C_{abio,i}}{df_{abio,i}} - \frac{C_{thresh,w^*i}}{df{abio,i}}) \ge 0 \
0 & \textrm{otherwise}
\end{cases}
$$
Estimates for the abiotic clearance precision (abio.obs.prec):
$$ \tau_{abio,i} = \frac{1}{(\sigma_{a,w^i} + m{h,w^i} * x{abio,i})^2} $$
Likelihood for the abiotic clearance observations (abio.obs):
$$ y_{abio,i} \sim \textrm{N} \left( \mu = x_{abio,i},\tau = \tau_{abio,i} \right) $$
Post-MCMC Estimates
Conversion of disappearance rates (1/hr) to hepatic clearance ($\mu L/min/10^6$) for $1 \mu M$:
$$ \mathbf{Clint.1} = 1000\frac{m_{bio,1 \mu M}}{\rho_{hep}60} $$
where $\rho_{hep}$ is the hepatic density and $m_{bio,1 \mu M}$ is the slope for "bio" at $1 \mu M$ (i.e. $m_{bio,1} = m_{bio,1 \mu M}$ where $m_{bio,1}$ is the notation in earlier equations in the Clearance Model section).
Conversion of disappearance rates (1/hr) to hepatic clearance ($\mu L/min/10^6$) for $10 \mu M$:
$$ \mathbf{Clint.10} = 1000\frac{m_{bio,10 \mu M}}{\rho_{hep}60} $$
where $\rho_{hep}$ is the hepatic density and $m_{bio,10 \mu M}$ is the slope for "bio" at $10 \mu M$ (i.e. $m_{bio,2} = m_{bio,10 \mu M}$ where $m_{bio,2}$ is the notation in earlier equations in the Clearance Model section).
Posterior probability for intrinsic hepatic clearance:
$$ \mathbf{Clint.pValue} = \frac{\sum_{k=1}^{n_{M}} d_{c,k} }{n_{M}}$$
where $n_{M}$ is the number of MCMC iterations and $d_{c,k}$ is the binary result for MCMC iteration $k$ indicating whether the chemical decreases (i.e. is biologically cleared).
Posterior probability for the chemical saturation:
$$ \mathbf{Sat.pValue} = \frac{\sum_{k=1}^{n_{M}}s_{f,k}}{n_{M}} $$
where $n_{M}$ is the number of MCMC iterations and $s_{f,k}$ is the binary result for MCMC iteration $k$ indicating whether the saturation is hit in each of the MCMC iterations.
Posterior probability of the chemical degradation:
$$ \mathbf{degrades.pValue} = \frac{\sum_{k=1}^{n_{M}}d_{g,k}}{n_{M}}$$
where $n_{M}$ is the number of MCMC iterations and $d_{g,k}$ is the binary result for MCMC iteration $k$ indicating whether the chemical degradation is hit (i.e. is non-biologically cleared).
JAGS to L4 Equation Notation Tables
Data passed to JAGS as part of the PREJAGS object:
| Data | Notation | Description |
|-------------------|-----------------|-----------------|
| Test.Nominal.Conc | $conc_{target}$ | expected initial concentration |
| Num.cal | $n_{cal}$ | total number of calibrations |
| Num.obs | $n$ | total number of Cvst samples |
| obs | $y$ | Cvst sample responses |
| obs.conc | $C$ | Test.Nominal.Conc indices for Cvst samples |
| obs.time | $t$ | time the Cvst samples were tested for a given chemical concentration (in hours) |
| obs.cal | $w^$ | calibration index for Cvst samples (reused index) |
| obs.Dilution.Factor | $df$ | dilution factors for Cvst samples |
| Num.blank.obs | $n_{blank}$ | total number of Blank samples |
| Blank.obs | $y_{blank}$ | Blank sample responses |
| Blank.cal | $w^$ | calibration index for Blank samples (reused index) |
| Blank.Dilution.Factor | $df_{blank}$ | dilution factors for Blank samples |
| Num.cc | $n_{cc}$ | total number of CC samples |
| cc.obs.conc | $C_{cc}$ | Test.Compound.Conc for CC samples of the tested compounds |
| cc.obs | $y_{cc}$ | CC sample responses |
| cc.obs.cal | $w^$ | calibration index for CC samples (reused index) |
| cc.obs.Dilution.Factor | $df_{cc}$ | dilution factors for CC samples |
| Num.abio.obs | $n_{abio}$ | total number of Inactive samples |
| abio.obs | $y_{abio}$ | Inactive sample responses |
| abio.obs.conc | $C_{abio}$ | Test.Nominal.Conc indices for Inactive samples |
| abio.obs.time | $t_{abio}$ | time the Inactive samples were tested for a given chemical concentration (in hours) |
| abio.obs.cal | $w^$ | calibration index for Inactive samples (reused index) |
| abio.obs.Dilution.Factor | $df_{abio}$ | dilution factors for Inactive samples |
User-specified prior parameter values via level-4 function arguments and passed to JAGS as part of the PREJAGS object:
| Prior | Notation | level-4 Argument (Default) | Description |
|-----------|----------|------------------|-------------|
| DECREASE.PROB | $p_{d_c}$ | decrease.prob (0.5) | probability that a chemical will decrease in the assay |
| DEGRADE.PROB | $p_{d_g}$ | degrade.prob (0.05) | probability of instability for a chemical within the assay (i.e. abiotic degradation) |
| SATURATE.PROB | $p_{s_f}$ | saturate.prob (0.25) | probability that a chemical's rate of metabolism will decrease between the lower and higher concentration |
MCMC Parameters in JAGS:
| JAGS Parameter Name | Parameter | Distribution | Prior/Posterior/Calculated |
|---------------------|-----------|--------------|----------------------------|
| log.const.analytic.sd | $log(\sigma_a)$ | Uniform | Prior |
| log.hetero.analytic.slope | $log(m_h)$ | Uniform | Prior |
| C.thresh | $C_{thresh}$| Uniform | Prior |
| log.calibration | $log(cal)$ | Normal | Prior |
| background | $\gamma$ | Exponential | Prior |
| const.analytic.sd | $\sigma_a$ | | Calculated |
| hetero.analytic.slope | $m_h$ | | Calculated |
| calibration | $cal$ | | Calculated |
| Blank.pred | $x_{blank}$ | | Calculated |
| Blank.prec | $\tau_{blank}$ | | Calculated |
| Blank.obs | $y_{blank}$ | Normal | Posterior |
| cc.slope | $m_{cc}$ | | Calculated |
| cc.intercept | $\alpha_{cc}$ | | Calculated |
| cc.pred | $x_{cc}$ | | Calculated |
| cc.prec | $\tau_{cc}$ | | Calculated |
| cc.obs | $y_{cc}$ | Normal | Posterior |
| decreases | $d_c$ | Bernoulli | Prior |
| degrades | $d_g$ | Bernoulli | Prior |
| bio.rate | $\delta_{bio}$ | Uniform | Prior |
| abio.rate | $\delta_{abio}$ | Uniform | Prior |
| slope | $m$ | | Calculated |
| bio.slope | $m_{bio}$| | Calculated |
| saturates | $s_f$ | Bernoulli | Prior |
| saturation | $s_a$ | Uniform | Prior |
| C | $C$ | | Calculated |
| obs.pred | $x$ | | Calculated |
| obs.prec | $\tau$ | | Calculated (Not the general distribution parameter.) |
| obs | $y$ | Normal | Posterior |
| abio.slope | $m_{abio}$ | | Calculated |
| abio.C | $C_{abio}$ | | Calculated |
| abio.obs.pred | $x_{abio}$ | | Calculated |
| abio.obs.prec | $\tau_{abio}$ | | Calculated |
| abio.obs | $y_{abio}$ | Normal | Posterior |
References
\references{
\insertRef{breen2021high}{invitroTKstats}
\insertRef{shibata2002prediction}{invitroTKstats}
}
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```r knitr::opts_chunk$set( collapse = TRUE, comment = "#>", fig.align = 'center' )
Introduction
This vignette guides users on how to estimate intrinsic hepatic clearance (Cl~int~) from mass spectrometry data. Intrinsic hepatic clearance is a chemical specific parameter that describes a liver's ability to metabolize an unbound compound. It is characterized by the disappearance of compound when incubated with primary hepatocytes (@breen2021high).
The mass spectrometry data should be collected from an assay that uses cryopreserved hepatocyte suspension (@shibata2002prediction) as seen in Figure 1.
knitr::include_graphics("img/clint_diagram.png")
Suggested packages for use with this vignette
# Primary package library(invitroTKstats) # Data manipulation package library(dplyr) # Table formatting package library(flextable)
Load Data
First, we load in the example dataset from invitroTKstats.
# Load example clint data data("Clint-example")
Many datasets are loaded in: clint_L0, clint_L1,clint_L2, clint_L3, and clint_L4. These datasets are Cl~int~ data at Level 0, 1, 2, 3, and 4 respectively. Additional datasets associated with Level 4 processing are also loaded in: clint_L2_heldout and clint_PREJAGS. These will be described later in the "Level 4 processing" section.
Lastly, a clint_cheminfo dataset is loaded in that contains chemical information necessary for identification mapping; it is used to create Level 0 data. For the purpose of this vignette, we'll start with clint_L0, the Level 0 data, to demonstrate the complete pipelining process.
clint_L0 is the output from the merge_level0 function which compiles raw lab data from specified Excel files into a singular data frame. The data frame contains exactly one row per sample with information obtained from the mass spectrometer. For more details on curating raw lab data to a singular Level 0 data frame, see the "Data Guide Creation and Level-0 Data Compilation" vignette.
The following table displays the first three rows of clint_L0, our Level 0 data.
head(clint_L0, n = 3) %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( ) ) ) %>% set_caption(caption = "Table 1: Level 0 data", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
Level 1 processing
format_clint is the Level 1 function used to create a standardized data frame. This level of processing is necessary because naming conventions or formatting can differ across data sets.
If the Level 0 data already contains the required column, then the existing column name can be specified. For example, clint_L0 already contains a column specifying the sample name called "Sample". However, the default column name for sample name is "Lab.Sample.Name". Therefore, we specify the correct column with sample.col = "Sample". In general, to specify an already existing column that differs from the default, the user must use the parameter with the .col suffix.
If the Level 0 data does not already contain the required column, then the entire column can be populated with a single value. For example, clint_L0 does not contain a column specifying biological replicates. Therefore, we populate the required column with biological.replicates = 1. In general, to specify a single value for an entire column, the user must use the parameter without the .col suffix.
Users should be mindful if they choose to specify a single value for all of their samples; they should verify this action is one they wish to take.
Some columns must be present in the Level 0 data while others can be filled with a single value. At minimum, the following columns must be present in the Level 0 data and specification with a single entry is not permitted: sample.col, compound.col, dtxsid.col, lab.compound.col, type.col, istd.col, and area.col.
If there is no additional note.col in the Level 0 data, users should use note.col = NULL to fill the column with "Note".
The rest of the following columns may either be specified from the Level 0 data or filled with a single value: date.col or date, density.col or density, cal.col or cal, dilution.col or dilution, time.col or time, istd.name.col or istd.name, istd.conc.col or istd.conc, test.conc.col or test.conc, test.nominal.conc.col or test.nominal.conc, biological.replicates.col or biological.replicates, analysis.method.col or analysis.method, analysis.instrument.col or analysis.instrument, analysis.parameters.col or analysis.parameters, level0.file.col or level0.file, and level0.sheet.col or level0.sheet.
# Create table of required parameters for Level 1 req_cols = data.frame(matrix(nrow = 31, ncol = 5)) vars <- c("Argument", "Default", "Required in L0?", "Corresp. single-entry Argument", "Descr.") colnames(req_cols) <- vars # Argument names arguments <- c("FILENAME", "data.in", "sample.col", "date.col", "compound.col", "dtxsid.col", "lab.compound.col", "type.col", "density.col", "cal.col", "dilution.col", "time.col", "istd.col", "istd.name.col", "istd.conc.col", "test.conc.col", "test.nominal.conc.col", "area.col", "biological.replicates.col", "technical.replicates.col", "analysis.method.col", "analysis.instrument.col", "analysis.parameters.col", "note.col", "level0.file.col", "level0.sheet.col", "output.res", "save.bad.types", "sig.figs", "INPUT.DIR", "OUTPUT.DIR") req_cols[,"Argument"] <- arguments # Default arguments defaults <- c("MYDATA", NA, "Lab.Sample.Name", "Date", "Compound.Name", "DTXSID", "Lab.Compound.Name", "Sample.Type", "Hep.Density", "Cal", "Dilution.Factor", "Time", "ISTD.Area", "ISTD.Name", "ISTD.Conc", "Test.Compound.Conc", "Test.Target.Conc", "Area", "Biological.Replicates", "Technical.Replicates", "Analysis.Method", "Analysis.Instrument", "Analysis.Parameters", "Note", "Level0.File", "Level0.Sheet", "FALSE", "FALSE", 5, NA, NA) req_cols[,"Default"] <- defaults # Argument required in L0? req_cols <- req_cols %>% mutate("Required in L0?" = case_when( Argument %in% c("sample.col","compound.col", "dtxsid.col", "lab.compound.col", "type.col", "istd.col", "area.col") ~ "Y", Argument %in% c("FILENAME", "data.in", "output.res", "save.bad.types", "sig.figs", "INPUT.DIR", "OUTPUT.DIR") ~ "N/A", .default = "N" )) # Corresponding single-entry Argument req_cols <- req_cols %>% mutate("Corresp. single-entry Argument" = ifelse(.data[[vars[[3]]]] == "N" & .data[[vars[[1]]]] != "note.col", gsub(".col","",Argument), NA)) # Brief description description <- c("Output and input filename", "Level 0 data frame", "Lab sample name", "Lab measurement date", "Formal test compound name", "EPA's DSSTox Structure ID", "Lab test compound name (abbr.)", "Sample type (Blank/Cvst/Inactive/CC)", "Hepatocyte density in the in vitro incubation", "MS calibration", "Number of times sample was diluted", "Time since chemical was introduced", "Internal standard peak area", "Internal standard name", "Internal standard concentration", "Standard test chemical concentration", "Initial chemical concentration", "Target analyte peak area", "Replicates with the same analyte", "Repeated measurements from one sample", "Analytical chemistry analysis method", "Analytical chemistry analysis instrument", "Analytical chemistry analysis parameters", "Additional notes", "Raw data filename", "Raw data sheet name", "Export results (TSV)?", "Export bad data (TSV)?", "Number of significant figures", "Input directory of Level 0 file", "Export directory to save Level 1 files" ) req_cols[,"Descr."] <- description
req_cols %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( height = 200 ) ) ) %>% set_caption(caption = "Table 2: Level 1 'format_clint' function arguments", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
A TSV file containing the level-1 data can be exported to the user's per-session temporary directory. This temporary directory is a per-session directory whose path can be found with the following code: tempdir(). For more details, see
[https://www.collinberke.com/til/posts/2023-10-24-temp-directories/].
To avoid exporting to this temporary directory, an OUTPUT.DIR must be specified. We have omitted this export entirely with output.res = FALSE (the default). The option to omit exporting a TSV file is also available at levels 2 and 3 and will be used from this point forward.
clint_L1_curated <- format_clint(FILENAME = "Clint_vignette", data.in = clint_L0, # columns present in L0 data sample.col = "Sample", compound.col = "Compound", lab.compound.col = "Lab.Compound.ID", type.col = "Type", istd.col = "ISTD.Peak.Area", area.col = "Peak.Area", note.col = "Sample Text", test.conc.col = "Compound.Conc", analysis.parameters.col = "Analysis.Params", # columns not present in L0 data density = 0.5, test.nominal.conc = 1, analysis.instrument = "Unknown", analysis.method = "LCMS", istd.conc = 0.01, cal = 1, biological.replicates = 1, # don't export output TSV file output.res = FALSE )
We receive a warning message that some of our samples have been removed due to "inappropriate sample types". The removed samples include data that did not have one of the following sample types
- Blank with no chemical added (Blank)
- Hepatocyte incubation concentration (Cvst)
- Inactivated Hepatocytes (Inactive)
- Calibration Curve (CC)
The following table displays some of the samples that were removed. These samples are annotated with a "QC" sample type indicating "Quality Control" and should therefore not be included in our analysis.
clint_L0 %>% filter(Type == "QC") %>% head(n = 5) %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( height = 200 ) ) ) %>% set_caption(caption = "Table 3: Subset of removed samples", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
Users can verify that these samples are excluded by filtering their Level 1 data frame and ensuring no matches are found. One approach is given below.
# All the samples of an inappropriate sample type excluded <- clint_L0 %>% filter(!Type %in% c("Blank", "Cvst", "Inactive", "CC")) # Exclude based on Sample and DTXSID X <- c(excluded$Sample) names(X) <- paste(excluded$Sample, excluded$DTXSID, sep = "+") Y <- c(excluded$DTXSID) # Find samples in L1 data frame with matching sample name (X) and DTXSID (Y) matches <- as.data.frame(t(mapply(function(X,Y) {subset(clint_L1_curated, Lab.Sample.Name == X & DTXSID == Y)}, X, Y, USE.NAMES = T))) matches # Check that no matches were returned for each sample in `excluded` check <- rep(1,nrow(matches)) names(check) <- rownames(matches) for (name in rownames(matches)) { # If no matches were found, each element of check should evaluate to 0 check[name] <- sum(sapply(c(1:ncol(matches)), function(X) {length(matches[1,X][[1]])})) } # Verify that each sample in `excluded` had no matches check # If no matches, should evaluate to 0 sum(check)
To export the removed samples types as a TSV, the user can set the parameter save.bad.types = TRUE. For this example, we will not save the discarded sample types.
The following table displays the first three rows of clint_L1_curated, our Level 1 data produced from format_clint. In addition to the columns specified by the user, there is an additional column called Response. This column is the test compound concentration and is calculated as $\textrm{Response} = \frac{\textrm{Analyte Area}}{\textrm{ISTD Area}}*\textrm{ISTD Conc}$ where $\textrm{Analyte Area}$ is defined by the Area column, $\textrm{ISTD Area}$ is defined by the ISTD.Area column, and $\textrm{ISTD Conc}$ is defined by the ISTD.Conc column.
clint_L1_curated %>% head(n = 3) %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( ) ) ) %>% set_caption(caption = "Table 4: Level 1 data", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
Level 2 processing
sample_verification is the Level 2 function used to add a verification column. The verification column indicates whether a sample should be included in the point estimation (Level 3) and credible interval (Level 4) processing. This column allows users to keep all samples in their data but only utilize the reliable samples for Cl~int~ estimation. All of the data in Level 2 is identical to the data in Level 1 with the exception of the additional Verified column.
To determine whether a sample should be included, the user should consult the wet-lab scientists from where their data originates or a chemist who may be able to provide reliable rationale for samples that should not be verified. This level of processing allows the user to receive feedback from the wet-lab scientists, exclude erroneous or unreliable samples, and produce new Cl~int~ estimates. Thus, there is always an open channel of communication between the user and the wet-lab scientists or chemists.
We will use the already processed Level 2 data frame, clint_L2, to regenerate our exclusion data. In general, the user would not have access to the exclusion information a priori.
The exclusion data frame must include the following columns: Variables, Values, and Message. The Variables column contains the variable names used to filter the excluded rows. Here, we are using Lab.Sample.Name and DTXSID to identify the excluded rows separated by a "|". The Values column contains the values of the variables, as a character, also separated by a "|". The Message column contains the reason for exclusion. Here, we are using the reasons listed in the Verified column in clint_L2. The user should refrain from using "|" in any of their descriptions to avoid conflicts with the sample_verification function.
# Use verification data from loaded in `clint_L2` data frame exclusion <- clint_L2 %>% filter(Verified %in% c("Wrong Mix", "Unknown Conc.")) %>% mutate("Variables" = "Lab.Sample.Name|DTXSID") %>% mutate("Values" = paste(Lab.Sample.Name, DTXSID, sep = "|")) %>% mutate("Message" = Verified) %>% select(Variables, Values, Message)
exclusion %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( height = 200 ) ) ) %>% set_caption(caption = "Table 5: Exclusion data frame", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
clint_L2_curated <- sample_verification(FILENAME = "Clint_vignette", data.in = clint_L1_curated, assay = "Clint", exclusion.info = exclusion, # don't export output TSV file output.res = FALSE)
Our Level 2 data now contains a Verified column. If the sample should be included, the column contains a "Y" for yes. If the sample should be excluded, the column contains the reason for exclusion.
The following table displays some rows of the Level 2 data, each with a different value in the Verification column. Note, the reasons for exclusion are not limited to "Unknown Conc." or "Wrong Mix", they were merely provided as an example.
df1 <- head(clint_L2_curated, n = 1) df2 <- head(filter(clint_L2_curated, Verified == "Wrong Mix"), n = 1) df3 <- head(filter(clint_L2_curated, Verified == "Unknown Conc."), n = 1) temp <- rbind(df1, df2) temp <- rbind(temp, df3)
temp %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( ) ) ) %>% set_caption(caption = "Table 6: Level 2 data", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
Level 3 processing
calc_clint_point is the Level 3 function used to calculate the Cl~int~ point estimate for each test compound using a Frequentist framework.
Mathematically, Cl~int~ is the slope of the line representing log concentrations over time. With some assumptions on the form of the line and on the distribution of the error terms, we can estimate Cl~int~ using maximum likelihood estimation.
First, we assume the test compound concentration at time $t$ is an exponential decay model $$C(t) = \textrm{cal}C_0e^{-mt}$$ where $\textrm{cal}$ is the MS calibration, $C_0$ is the test compound concentration at time 0, $m$ is the rate constant, and $t$ is the incubation time. Notice that if a natural log is applied, $$\textrm{ln}(C(t)) = -mt + \textrm{ln}(\textrm{cal}*C_0)$$ $m$ is the slope of the line representing log concentrations over time. Thus, the rate constant $m$ is our Cl~int~ value.
Next, we also assume the random noise is normally distributed $\varepsilon_i \sim \mathcal{N}(0,\sigma^2)$ where $\sigma^2$ is unknown.
Lastly, the test compound concentration, our dependent variable, is qualitatively defined by our Response column, the ratio of the analyte area to the ISTD area.
With this information, we can rewrite our dependent variable as $$y_i \sim \mathcal{N}(C(t),\sigma^2)$$ After some simplification, our log-likelihood is defined as $$l(\theta) = N*log\left(\frac{1}{\sqrt{2\pi}\sigma}\right)-\frac{1}{2}\sum_{i=1}^N\frac{(y_i-C(\theta, t))^2}{\sigma^2}$$ where $\theta = (\textrm{cal}, m)$ and $N$ is the number of Cvst and Blank samples.
Using the mle estimator in the stats4 package, we then estimate the parameters that maximize the log-likelihood for each test compound.
clint_L3_curated <- calc_clint_point(FILENAME = "Clint_vignette", data.in = clint_L2_curated, # don't export output TSV file output.res = FALSE)
Our Level 3 data contains a Clint estimate, Clint.pValue, AIC of the exponential decay fit, and AIC.Null of the exponential decay assuming a constant rate of decay.
Some experiments also include the assay performed at 1 $\mu M$ and 10 $\mu M$ to check for the saturation of metabolizing enzymes. The test compound concentration at time $t$ with a saturation probability is assumed to follow an exponential decay model with a slight modification $$C_{sat}(t) = \textrm{cal}C_0e^{-m\textrm{sat}t}$$ where $\textrm{cal}$ is the MS calibration, $C_0$ is the test compound concentration at time 0, $m$ is the rate constant, $t$ is the incubation time, and $\textrm{sat}$ is the probability of saturation, or the probability of observing a lower clearance rate at a higher concentration.
If these samples are included, Clint.1 is the clearance rate at 1 $\mu M$, Clint.10 is the clearance rate at 10 $\mu M$, AIC.Sat is the AIC of the exponential decay with a saturation probability, and Sat.pValue is the corresponding p-value.
Because our example data did not include these samples, Clint.1, Clint.10, AIC.Sat, and Sat.pValue are empty.
clint_L3_curated %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( ) ) ) %>% set_caption(caption = "Table 7: Level 3 data", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
Level 4 processing
calc_clint is the Level 4 function used to calculate Cl~int~ point estimates and credible intervals using a Bayesian framework. Markov chain Monte Carlo (MCMC) simulations are used to randomly sample from the posterior distribution with a uniform prior.
To run Level 4, one needs to have JAGS installed on their machine. To determine the correct path, the user may use the runjags::findjags() or may need to specify the JAGS installation location for the JAGS.PATH argument.
:::{.noticebox}
JAGS is a software used for conducting Bayesian hierarchical modeling using Markov Chain Monte Carlo (MCMC) simulation. invitroTKstats contains the JAGS models for each of the applicable assays and utilizes the JAGS model under the hood to run the MCMC simulations via the runjags dependency to obtain the level-4 Bayesian estimates.
- Download and install the "Just Another Gibbs Sampler" (JAGS) software, which is freely available here.
- Once JAGS is installed one will need to identify the installation location, which is likely to be in a system location (e.g. "Program Files"). Users may need this location to specify the
JAGS.PATHargument within the level-4 function. - Finally, if not already installed, the
rjags,runjags, andcodaR packages need to be installed via the R console using theinstall.packagesfunction.
:::
We pass clint_L2_curated, the Level 2 data frame, and not clint_L3_curated, the Level 3 data frame, into calc_clint. This is because Level 3 and Level 4 processing are not sequential; they are methods that calculate different statistical quantities. The following code chunk takes a while to run; previous runtimes are around 7 minutes.
clint_L4_curated <- calc_clint(FILENAME = "Clint_vignette", data.in = clint_L2_curated, JAGS.PATH = runjags::findjags() )
clint_L4_curated <- clint_L4
The Cl~int~ intervals are returned to the user's R session, in an exported TSV file, and in an exported RData file. There is no parameter to prevent the TSV or RData files from being exported because of the potential for the simulations to crash. If there are no crashes, then the exported TSV file is identical to the user's R session and the exported RData file. clint_L4 is an example exported RData file.
Additionally, intermediate files are saved to the user's current working directory if TEMP.DIR = NULL. These include a Level 2 heldout set, clint_L2_heldout, containing unverified samples and a Level 4 PREJAGS list, clint_PREJAGS, containing arguments provided to JAGS. Because the PREJAGS list is overwritten with each compound, clint_PREJAGS only contains information relevant to the last tested compound, TFMFPA in this case.
Our Level 4 data contains a Cl~int~ credible interval and the corresponding p-value for each chemical.
clint_L4_curated %>% flextable() %>% bg(bg = "#DDDDDD", part = "header") %>% autofit() %>% set_table_properties( opts_html = list( scroll = list( ) ) ) %>% set_caption(caption = "Table 8: Level 4 data", align_with_table = FALSE) %>% fontsize(size = 10, part = "all") %>% theme_vanilla()
Best Practices: Food for thought
Generally, data processing pipelines should include minimal to no manual coding. It is best to keep clean code that is easily reproducible and transferable. The user should aim to have all the required data and meta-data files properly formatted to avoid further modifications throughout the pipeline.
Appendix
L4 Bayesian Modeling Information
In this section, we provide the equations for the Bayesian model (i.e., priors and likelihoods) used to estimate the intrinsic hepatic clearance ($Cl_{int}$) and the uncertainty about that estimate. The following sub-sections are organized such that:
- Each section contains the relevant hyper-parameters and their priors followed by the variable likelihoods
- Sections are ordered from the most fundamental variables/parameters (i.e. those used in later equations) to the likelihood for the observations used to estimate the intrinsic hepatic clearance ($Cl_{int}$)
Some of the indices are reused between sections (e.g. $i$, $w^*_i$, etc.). However, it should be noted that these are not meant to be understood across sub-sections; rather, only understood within the context of the section they are in.
NOTE: Readers unfamiliar with JAGS should be aware that JAGS software uses precision ($\tau$) rather than variance ($\sigma^2$) (i.e., $\tau = \frac{1}{\sigma^2}$).
Measurement Model
Each chemical may have more than one day of experimentation, and thus multiple calibrations. Suppose for the chemical of interest there are a total of $n_{cal}$ calibrations (Num.cal). For a particular calibration $w \in (1,\ldots,n_{cal})$ we assume the following priors for our Bayesian model:
Prior for log-scale constant analytic standard deviation (log.const.analytic.sd):
$$ log(\sigma_a)w \sim \textrm{Unif} \left( a = -6,b = 1 \right) ; \space \sigma{a,w} = 10^{log(\sigma_a)_w} $$
where $a$ and $b$ are the minimum and maximum, respectively, and $\sigma_{a,w}$ is the converted parameter used in later equations (const.analytic.sd).
Prior for the log-scale heteroscedastic analytic slope (log.hetero.analytic.slope):
$$ log(m_h)w \sim \textrm{Unif} \left( a = -6,b = 1 \right) ; \space m{h,w} = 10^{log(m_h)_w} $$
where $a$ and $b$ are the minimum and maximum, respectively, and $m_{h,w}$ is the converted parameter used in later equations (hetero.analytic.slope).
Prior for the threshold concentration (C.thresh):
$$ C_{thresh,w} \sim \textrm{Unif} \left( a = 0,b = \frac{conc_{target,w}}{10} \right)$$
where $a$ and $b$ are the minimum and maximum, respectively, and $conc_{target,w}$ is the expected initial concentration (Test.Nominal.Conc) for calibration index $w$.
Prior for the log-scale calibration (log.calibration):
$$ log(cal)_w \sim \textrm{N} \left( \mu = 0,\tau = 0.01 \right) ; \space cal_w = 10^{log(cal)_w}$$
where $\mu$ and $\tau$ are the mean and precision, respectively, and $cal_w$ is the converted parameter used in later equations (calibration).
Prior for the background (background):
$$ \gamma_w \sim \textrm{Exp} \left( \lambda = 100 \right)$$
where $\lambda$ is the rate parameter.
Estimation of Blanks
Let us assume the total number of prediction and precision for the blank observations is based on the total number of calibrations in the set (i.e. $n_{cal}$, Num.cal). Thus, for each calibration $j \in w$ we obtain the following:
Estimation for the predicted values for blank observations (Blank.pred):
$$ x_{blank,j} = \frac{\gamma_{j}}{df_{blank,j}} $$
where $df_{blank,j}$ is the dilution factor for the $j^{th}$ calibration for blank observations (Blank.Dilution.Factor).
Estimation for the precision of blank observations (Blank.prec):
$$ \tau_{blank,j} = \frac{1}{(\sigma_{a,j} + m_{h,j} *x_{blank,j})^2} $$
Likelihood for blank observations (Blank.obs):
Suppose $n_{blank}$ is the total number of blank observations (Num.blank.obs), and $y_{blank,i}$ indicates the $i^{th}$ blank observation, sample type = "Blank" (Blank.obs). For each observation we obtain a posterior MCMC estimate for the blank observations with the following:
$$ y_{blank,i} \sim \textrm{N} \left( \mu = x_{blank,w^i}, \tau = \tau{blank,w^_i} \right) $$
where $w^_i$ is the calibration index (Blank.cal) for the $i^{th}$ observation, such that $w^ = (w^_1,\ldots,w^{n{blank}})$ and $w^_i \in w$ (i.e. $w^i$ indicates one of the $n{cal}$ calibrations).
Estimation of Calibration Curve
Suppose $n_{cc}$ is the total number of calibration curve observations (Num.cc), and $y_{cc,i}$ indicates the $i^{th}$ calibration curve observation, sample type = CC (cc.obs). For each observation we obtain a posterior MCMC estimate for the calibration curve observations with the following:
Calibration curve slope for the $i^{th}$ observation (cc.slope) is assumed to be the calibration estimate corresponding to the calibration index ($w^*_i$):
$$ m_{cc,i} = cal_{w^*_i}$$
where $w^_i$ is the calibration index (cc.obs.cal) for the $i^{th}$ observation, such that $w^ = (w^_1,\ldots,w^{n{cc}})$ and $w^_i \in w$ (i.e. $w^i$ indicates one of the $n{cal}$ calibrations).
Calibration curve intercept for the $i^{th}$ observation (cc.intercept) is assumed to be the background estimate corresponding to the calibration index ($w^*_i$):
$$ \alpha_{cc,i} = \gamma_{w^*_i}$$
Estimation of the predictions for calibration curve observations (cc.pred):
$$ x_{cc,i} = m_{cc,i} * (\frac{C_{cc,i}}{df_{cc,i}} - \frac{C_{thresh,w^*i}}{df{cc,i}}) * \beta_{cc,i} + \frac{\alpha_{cc,i}}{df_{cc,i}} $$
where
- $C_{cc,i}$ is the chemical concentration (
cc.obs.conc) for the $i^{th}$ calibration curve observation - $df_{cc,i}$ is the dilution factor (
cc.obs.Dilution.Factor) for the $i^{th}$ calibration curve observation - $\beta_{cc,i}$ is the JAGS step function for the $i^{th}$ calibration curve observation, such that
$$ \beta_{cc,i} = \begin{cases} 1 & \textrm{if} \space (\frac{C_{cc,i}}{df_{cc,i}} - \frac{C_{thresh,w^*i}}{df{cc,i}}) \ge 0 \ 0 & \textrm{otherwise} \end{cases} $$
Estimation of the precision for calibration curve observations (cc.prec):
$$ \tau_{cc,i} = \frac{1}{(\sigma_{a,w^i} + m{h,w^i} * x{cc,i})^2} $$
Likelihood for the calibration curve observations (cc.obs):
$$ y_{cc,i} \sim \textrm{N} \left( \mu = x_{cc,i},\tau = \tau_{cc,i} \right)$$
Clearance Model
In some cases, the intrinsic hepatic clearance assay will have two initial concentrations (i.e. lower and higher concentration), typically these are $1$ and $10 \mu M$ but do not necessarily need to be.
Prior for the binary indicator for whether the concentration decreases (decreases):
$$ d_c \sim \textrm{Bern} \left( p = p_{d_c} \right) $$
where $p$ is the probability parameter and $p_{d_c}$ is the user-specified prior probability that a chemical will decrease in the assay (DECREASE.PROB).
Prior for the binary indicator for whether the concentration degrades (degrades):
$$ d_g \sim \textrm{Bern} \left( p = p_{d_g} \right) $$
where $p$ is the probability parameter and $p_{d_g}$ is the user-specified prior probability of instability for a chemical within the assay (i.e. abiotic degradation) (DEGRADE.PROB).
Prior for the slope representing the biological clearance rate (bio.rate) for the lower concentration, indicated by $C_{thresh,1}$ and $conc_{target,1}$:
$$ \delta_{bio} \sim \textrm{Unif} \left( a = 0,b = -log(\frac{C_{thresh,1}}{conc_{target,1}}) \right)$$
where $a$ and $b$ are the minimum and maximum, respectively.
Prior for the slope representing the abiotic clearance rate, when data from the inactivated hepatocytes is available (abio.rate) at the lower concentration, indicated by $C_{thresh,1}$ and $conc_{target,1}$:
$$ \delta_{abio} \sim \textrm{Unif} \left( a = 0,b = -log(\frac{C_{thresh,1}}{conc_{target,1}}) \right) $$
where $a$ and $b$ are the minimum and maximum, respectively.
Prior for the binary indicator for whether there is a reduction in the biological clearance and the chemical shows some metabolic saturation between the lower and higher concentration (saturates):
$$ s_f \sim \textrm{Bern} \left( p = p_{s_f} \right)$$
where $p$ is the probability parameter and $p_{s_f}$ is the user-specified prior probability that a chemical's rate of metabolism will decrease between the lower and higher concentration (SATURATE.PROB).
$p_{s_f} = 0.25$ assumes $25\%$ of the chemical shows some metabolic saturation between the lower and higher concentrations.
Prior for the saturation at the higher concentration (i.e. magnitude of decreased biological clearance) at the higher concentration (saturation):
$$ s_a \sim \textrm{Unif} \left( a = 0,b = 1 \right) $$
Estimation of the actual biological clearance rate (bio.slope) at the lower ($m_{bio,1}$) and higher ($m_{bio,2}$) initial concentrations, respectively:
$$ m_{bio,1} = d_c * \delta_{bio} $$ $$ m_{bio,2} = d_c * \delta_{bio} (1-s_fs_a) $$
such that $m_{bio} = (m_{bio,1},m_{bio,2})$.
The total elimination rate (slope) at the lower ($m_1$) and higher ($m_2$) initial concentrations, respectively:
$$ m_1 = d_c * \delta_{bio} + d_g * \delta_{abio} $$
$$ m_2 = d_g * \delta_{abio} + d_c * \delta_{bio} (1-s_fs_a) $$
such that $m = (m_1,m_2)$.
Estimate of the abiotic clearance rate (abio.slope):
$$ m_{abio} = d_g * \delta_{abio} $$
Estimation of Metabolic Clearance
Suppose $n$ is the total number of observations (Num.obs), and $y_{i}$ indicates the $i^{th}$ observation, sample type = Cvst (obs). For each observation we obtain a posterior MCMC estimate for the calibration curve observations with the following:
Estimate of the metabolic exponential decay for the $i^{th}$ observation (C):
$$ C_i = conc_{target,c^_i}exp(-m_{c^_i}t_i) $$
where
- $c^_i$ represents the initial concentration index (
obs.conc) for the $i^{th}$ observation (i.e, $c^ = (c^_1,\ldots,c^_n)$) and $c^*_i \in (1,2)$ such that $1$ indicates the lower initial concentration and $2$ indicates the higher initial concentration - $conc_{target,c^_i}$ is the expected initial concentration (
Test.Nominal.Conc) corresponding to the $i^{th}$ observation, given the concentration index $c^_i$ - $m_{c^i}$ is the total elimination rate slope for the initial concentration corresponding to the $i^{th}$ observation and $m{c^_i} \in m$
- $t_i$ is the time (
obs.time) for the $i^{th}$ observation
Estimate of the metabolic clearance predictions (obs.pred):
$$ x_i = cal_{w^i} * (\frac{C_i}{df_i} - \frac{C{thresh,w^i}}{df_i}) * \beta_i + \frac{\gamma{w^*_i}}{df_i} $$
where
- $df_i$ is the dilution factor (
obs.Dilution.Factor) for the $i^{th}$ observation - $\beta_i$ is the JAGS step function for the $i^{th}$ observation, such that
$$ \beta_i = \begin{cases} 1 & \textrm{if} \space (\frac{C_i}{df_i} - \frac{C_{thresh,w^*_i}}{df_i}) \ge 0 \ 0 & \textrm{otherwise} \end{cases} $$
- $w^_i$ represents the calibration index (
obs.cal) for the $i^{th}$ observation $w^ = (w^_1,\ldots,w^{n})$ and $w^_i \in w$ (i.e. $w^_i$ indicates one of the $n{cal}$ calibrations)
Estimates of the metabolic clearance precision (obs.prec):
$$ \tau_i = \frac{1}{(\sigma_{a,w^i} + m{h,w^_i}*x_i)^2} $$
Likelihood for metabolic clearance (obs):
$$ y_i \sim \textrm{N} \left( \mu = x_i,\tau = \tau_i \right)$$
Estimation of Abiotic Clearance
Suppose $n_{abio}$ is the total number of abiotic observations (Num.abio.obs), and $y_{abio,i}$ indicates the $i^{th}$ observation, sample type = Inactive (abio.obs). For each observation we obtain a posterior MCMC estimate for the calibration curve observations with the following:
Estimate of the abiotic exponential decay for the $i^{th}$ observation (abio.C):
$$ C_{abio,i} = conc_{target,w^i} * exp(-m{abio}t_{abio,i}) $$
- $w^_i$ represents the calibration index (
abio.obs.cal) for the $i^{th}$ non-biological observation $w^ = (w^_1,\ldots,w^{n_abio})$ and $w^_i \in w$ (i.e. $w^_i$ indicates one of the $n{cal}$ calibrations) - $conc_{target,w^_i}$ is the expected initial concentration (
Test.Nominal.Conc) corresponding to the $i^{th}$ observation, given the calibration index $w^_i$ - $t_{abio,i}$ is the time for the $i^{th}$ abiotic observation (
abio.obs.time)
Estimates for the abiotic clearance predictions (abio.obs.pred):
$$ x_{abio,i} = cal_{w^i} * (\frac{C{abio,i}}{df_{abio,i}} - \frac{C_{thresh,w^i}}{df{abio,i}}) * \beta_{abio,i} + \frac{\gamma_{w^*i}}{df{abio,i}} $$
where
- $df_{abio,i}$ is the dilution factor (
abio.obs.Dilution.Factor) for the $i^{th}$ abiotic observation - $\beta_{abio,i}$ is the JAGS step function for the $i^{th}$ abiotic observation, such that
$$ \beta_{abio,i} = \begin{cases} 1 & \textrm{if} \space (\frac{C_{abio,i}}{df_{abio,i}} - \frac{C_{thresh,w^*i}}{df{abio,i}}) \ge 0 \ 0 & \textrm{otherwise} \end{cases} $$
Estimates for the abiotic clearance precision (abio.obs.prec):
$$ \tau_{abio,i} = \frac{1}{(\sigma_{a,w^i} + m{h,w^i} * x{abio,i})^2} $$
Likelihood for the abiotic clearance observations (abio.obs):
$$ y_{abio,i} \sim \textrm{N} \left( \mu = x_{abio,i},\tau = \tau_{abio,i} \right) $$
Post-MCMC Estimates
Conversion of disappearance rates (1/hr) to hepatic clearance ($\mu L/min/10^6$) for $1 \mu M$:
$$ \mathbf{Clint.1} = 1000\frac{m_{bio,1 \mu M}}{\rho_{hep}60} $$
where $\rho_{hep}$ is the hepatic density and $m_{bio,1 \mu M}$ is the slope for "bio" at $1 \mu M$ (i.e. $m_{bio,1} = m_{bio,1 \mu M}$ where $m_{bio,1}$ is the notation in earlier equations in the Clearance Model section).
Conversion of disappearance rates (1/hr) to hepatic clearance ($\mu L/min/10^6$) for $10 \mu M$:
$$ \mathbf{Clint.10} = 1000\frac{m_{bio,10 \mu M}}{\rho_{hep}60} $$
where $\rho_{hep}$ is the hepatic density and $m_{bio,10 \mu M}$ is the slope for "bio" at $10 \mu M$ (i.e. $m_{bio,2} = m_{bio,10 \mu M}$ where $m_{bio,2}$ is the notation in earlier equations in the Clearance Model section).
Posterior probability for intrinsic hepatic clearance:
$$ \mathbf{Clint.pValue} = \frac{\sum_{k=1}^{n_{M}} d_{c,k} }{n_{M}}$$
where $n_{M}$ is the number of MCMC iterations and $d_{c,k}$ is the binary result for MCMC iteration $k$ indicating whether the chemical decreases (i.e. is biologically cleared).
Posterior probability for the chemical saturation:
$$ \mathbf{Sat.pValue} = \frac{\sum_{k=1}^{n_{M}}s_{f,k}}{n_{M}} $$
where $n_{M}$ is the number of MCMC iterations and $s_{f,k}$ is the binary result for MCMC iteration $k$ indicating whether the saturation is hit in each of the MCMC iterations.
Posterior probability of the chemical degradation:
$$ \mathbf{degrades.pValue} = \frac{\sum_{k=1}^{n_{M}}d_{g,k}}{n_{M}}$$
where $n_{M}$ is the number of MCMC iterations and $d_{g,k}$ is the binary result for MCMC iteration $k$ indicating whether the chemical degradation is hit (i.e. is non-biologically cleared).
JAGS to L4 Equation Notation Tables
Data passed to JAGS as part of the PREJAGS object:
| Data | Notation | Description |
|-------------------|-----------------|-----------------|
| Test.Nominal.Conc | $conc_{target}$ | expected initial concentration |
| Num.cal | $n_{cal}$ | total number of calibrations |
| Num.obs | $n$ | total number of Cvst samples |
| obs | $y$ | Cvst sample responses |
| obs.conc | $C$ | Test.Nominal.Conc indices for Cvst samples |
| obs.time | $t$ | time the Cvst samples were tested for a given chemical concentration (in hours) |
| obs.cal | $w^$ | calibration index for Cvst samples (reused index) |
| obs.Dilution.Factor | $df$ | dilution factors for Cvst samples |
| Num.blank.obs | $n_{blank}$ | total number of Blank samples |
| Blank.obs | $y_{blank}$ | Blank sample responses |
| Blank.cal | $w^$ | calibration index for Blank samples (reused index) |
| Blank.Dilution.Factor | $df_{blank}$ | dilution factors for Blank samples |
| Num.cc | $n_{cc}$ | total number of CC samples |
| cc.obs.conc | $C_{cc}$ | Test.Compound.Conc for CC samples of the tested compounds |
| cc.obs | $y_{cc}$ | CC sample responses |
| cc.obs.cal | $w^$ | calibration index for CC samples (reused index) |
| cc.obs.Dilution.Factor | $df_{cc}$ | dilution factors for CC samples |
| Num.abio.obs | $n_{abio}$ | total number of Inactive samples |
| abio.obs | $y_{abio}$ | Inactive sample responses |
| abio.obs.conc | $C_{abio}$ | Test.Nominal.Conc indices for Inactive samples |
| abio.obs.time | $t_{abio}$ | time the Inactive samples were tested for a given chemical concentration (in hours) |
| abio.obs.cal | $w^$ | calibration index for Inactive samples (reused index) |
| abio.obs.Dilution.Factor | $df_{abio}$ | dilution factors for Inactive samples |
User-specified prior parameter values via level-4 function arguments and passed to JAGS as part of the PREJAGS object:
| Prior | Notation | level-4 Argument (Default) | Description |
|-----------|----------|------------------|-------------|
| DECREASE.PROB | $p_{d_c}$ | decrease.prob (0.5) | probability that a chemical will decrease in the assay |
| DEGRADE.PROB | $p_{d_g}$ | degrade.prob (0.05) | probability of instability for a chemical within the assay (i.e. abiotic degradation) |
| SATURATE.PROB | $p_{s_f}$ | saturate.prob (0.25) | probability that a chemical's rate of metabolism will decrease between the lower and higher concentration |
MCMC Parameters in JAGS:
| JAGS Parameter Name | Parameter | Distribution | Prior/Posterior/Calculated |
|---------------------|-----------|--------------|----------------------------|
| log.const.analytic.sd | $log(\sigma_a)$ | Uniform | Prior |
| log.hetero.analytic.slope | $log(m_h)$ | Uniform | Prior |
| C.thresh | $C_{thresh}$| Uniform | Prior |
| log.calibration | $log(cal)$ | Normal | Prior |
| background | $\gamma$ | Exponential | Prior |
| const.analytic.sd | $\sigma_a$ | | Calculated |
| hetero.analytic.slope | $m_h$ | | Calculated |
| calibration | $cal$ | | Calculated |
| Blank.pred | $x_{blank}$ | | Calculated |
| Blank.prec | $\tau_{blank}$ | | Calculated |
| Blank.obs | $y_{blank}$ | Normal | Posterior |
| cc.slope | $m_{cc}$ | | Calculated |
| cc.intercept | $\alpha_{cc}$ | | Calculated |
| cc.pred | $x_{cc}$ | | Calculated |
| cc.prec | $\tau_{cc}$ | | Calculated |
| cc.obs | $y_{cc}$ | Normal | Posterior |
| decreases | $d_c$ | Bernoulli | Prior |
| degrades | $d_g$ | Bernoulli | Prior |
| bio.rate | $\delta_{bio}$ | Uniform | Prior |
| abio.rate | $\delta_{abio}$ | Uniform | Prior |
| slope | $m$ | | Calculated |
| bio.slope | $m_{bio}$| | Calculated |
| saturates | $s_f$ | Bernoulli | Prior |
| saturation | $s_a$ | Uniform | Prior |
| C | $C$ | | Calculated |
| obs.pred | $x$ | | Calculated |
| obs.prec | $\tau$ | | Calculated (Not the general distribution parameter.) |
| obs | $y$ | Normal | Posterior |
| abio.slope | $m_{abio}$ | | Calculated |
| abio.C | $C_{abio}$ | | Calculated |
| abio.obs.pred | $x_{abio}$ | | Calculated |
| abio.obs.prec | $\tau_{abio}$ | | Calculated |
| abio.obs | $y_{abio}$ | Normal | Posterior |
References
\references{ \insertRef{breen2021high}{invitroTKstats} \insertRef{shibata2002prediction}{invitroTKstats} }
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