# Calculate a Point Estimate of Apparent Membrane Permeability (Papp) from Caco-2 data (Level-3) This function calculates a point estimate of apparent membrane permeability (Papp) using mass spectrometry (MS) peak areas from samples collected as part of in vitro measurements of membrane permeability using Caco-2 cells \insertCite hubatsch2007determinationinvitroTKstats. ```r calc_caco2_point( FILENAME, data.in, good.col = "Verified", output.res = FALSE, sig.figs = 3, INPUT.DIR = NULL, OUTPUT.DIR = NULL, verbose = TRUE ) ``` ## Arguments - `FILENAME`: (Character) A string used to identify the input level-2 file, "-Caco-2-Level2.tsv" (if importing from a .tsv file), and/or used to identify the output level-3 file, "-Caco-2-Level3.tsv" (if exporting). - `data.in`: (Data Frame) A level-2 data frame generated from the `format_caco2` function with a verification column added by `sample_verification`. Complement with manual verification if needed. - `good.col`: (Character) Column name indicating which rows have been verified, data rows valid for analysis are indicated with a "Y". (Defaults to "Verified".) - `output.res`: (Logical) When set to `TRUE`, the result table (level-3) will be exported to the user's per-session temporary directory or `OUTPUT.DIR` (if specified) as a .tsv file. (Defaults to `FALSE`.) - `sig.figs`: (Numeric) The number of significant figures to round the exported result table (level-3). (Note: console print statements are also rounded to specified significant figures.) (Defaults to `3`.) - `INPUT.DIR`: (Character) Path to the directory where the input level-2 file exists. If `NULL`, looking for the input level-2 file in the current working directory. (Defaults to `NULL`.) - `OUTPUT.DIR`: (Character) Path to the directory to save the output file. If `NULL`, the output file will be saved to the user's per-session temporary directory or `INPUT.DIR` if specified. (Defaults to `NULL`.) - `verbose`: (**logical**) Indicate whether printed statements should be shown. (Default is TRUE.) ## Returns - **data.frame**: A level-3 data.frame in standardized format |||| |--:|--:|--:| |C0_A2B|Time zero donor concentration|Mass Spec Response Ratio (RR)| |dQdt_A2B|Estimated rate of mass movement through membrane|RR*cm^3/s| |Papp_A2B|Apparent membrane permeability|10^-6 cm/s| |C0_B2A|Time zero donor concentration|Mass Spec Response Ratio (RR)| |dQdt_B2A|Estimated rate of mass movement through membrane|RR*cm^3/s| |Papp_B2A|Apparent membrane permeability|10^-6 cm/s| |Refflux|Efflux ratio|unitless| |Frec_A2B.vec|Fraction recovered for the apical-basolateral direction, calculated as the fraction of the initial donor amount recovered in the receiver compartment (collapsed numeric vector, values for replicates separated by a "\|")|unitless| |Frec_A2B.mean|Mean of the fraction recovered for the apical-basolateral direction|unitless| |Frec_B2A.vec|Fraction recovered for the basolateral-apical direction, calculated in the same way as Frec_A2B.vec but in the opposite transport direction (collapsed numeric vector, values for replicates separated by a "\|")|unitless| |Frec_B2A.mean|Mean of the fraction recovered for the basolateral-apical direction|unitless| |Recovery_Class_A2B.vec|Recovery classification for apical-to-basolateral permeability("Low Recovery" if Frec_A2B.vec < 0.4 or "High Recovery" if Frec_A2B.vec > 2.0) (collapsed character vector, values for replicates separated by a "\|")|qualitative category| |Recovery_Class_A2B.mean|Recovery classification for the mean apical-to-basolateral permeability("Low Recovery" if Frec_A2B.mean < 0.4 or "High Recovery" if Frec_A2B.mean > 2.0)|qualitative category| |Recovery_Class_B2A.vec|Recovery classification for basolateral-to-apical permeability("Low Recovery" if Frec_B2A.vec < 0.4 or "High Recovery" if Frec_B2A.vec > 2.0) (collapsed character vector, values for replicates separated by a "\|")|qualitative category| |Recovery_Class_B2A.mean|Recovery classification for the mean basolateral-to-apical permeability("Low Recovery" if Frec_B2A.mean < 0.4 or "High Recovery" if Frec_B2A.mean > 2.0)|qualitative category| ## Details The input to this function should be "level-2" data. Level-2 data is level-1, data formatted with the `format_caco2` function, and curated with a verification column. "Y" in the verification column indicates the data row is valid for analysis. The data frame of observations should be annotated according to direction (either apical to basolateral -- "AtoB" -- or basolateral to apical -- "BtoA") and type of concentration measured: ||| |--:|--:| |Blank with no chemical added|Blank| |Target concentration added to donor compartment at time 0 (C0)|D0| |Donor compartment at end of experiment|D2| |Receiver compartment at end of experiment|R2| Apparent membrane permeability ($P_{app}$) is calculated from MS responses as: $P_{app} = \frac{dQ/dt}{c_0*A}$ The rate of permeation, $\frac{dQ}{dt}$$\left(\frac{\text{peak area}}{\text{time (s)}} \right)$ is calculated as: $\frac{dQ}{dt} = \max\left(0, \frac{\sum_{i=1}^{n_{R2}} (r_{R2} * c_{DF})}{n_{R2}} - \frac{\sum_{i=1}^{n_{BL}} (r_{BL} * c_{DF})}{n_{BL}}\right)$ where $r_{R2}$ is Receiver Response, $c_{DF}$ is the corresponding Dilution Factor, $r_{BL}$ is Blank Response, $n_{R2}$ is the number of Receiver Responses, and $n_{BL}$ is the number of Blank Responses. If the output level-3 result table is chosen to be exported and an output directory is not specified, it will be exported to the user's R 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/blog/posts/2023-10-24-til-temp-directories/](https://www.collinberke.com/blog/posts/2023-10-24-til-temp-directories/). As a best practice, `INPUT.DIR` (when importing a .tsv file) and/or `OUTPUT.DIR` should be specified to simplify the process of importing and exporting files. This practice ensures that the exported files can easily be found and will not be exported to a temporary directory. ## Examples ```r ## Load example level-2 data level2 <- invitroTKstats::caco2_L2 ## scenario 1: ## input level-2 data from the R session and do not export the result table level3 <- calc_caco2_point(data.in = level2, output.res = FALSE) ## scenario 2: ## import level-2 data from a 'tsv' file and export the result table to ## same location as INPUT.DIR ## Not run: ## Refer to sample_verification help file for how to export level-2 data to a directory. ## Unless a different path is specified in OUTPUT.DIR, ## the result table will be saved to the directory specified in INPUT.DIR. ## Will need to replace FILENAME and INPUT.DIR with name prefix and location of level-2 'tsv'. level3 <- calc_caco2_point(# e.g. replace with "Examples" from "Examples-Caco-2-Level2.tsv" FILENAME="", INPUT.DIR = "", output.res = TRUE) ## End(Not run) ## scenario 3: ## input level-2 data from the R session and export the result table to the ## user's temporary directory ## Will need to replace FILENAME with desired level-2 filename prefix. ## Not run: level3 <- calc_caco2_point(# e.g. replace with "MYDATA" FILENAME = "", data.in = level2, output.res = TRUE) # To delete, use the following code. For more details, see the link in the # "Details" section. file.remove(list.files(tempdir(), full.names = TRUE, pattern = "-Caco-2-Level3.tsv")) ## End(Not run) ``` ## References \insertRef hubatsch2007determinationinvitroTKstats ## Author(s) John Wambaugh

calc_caco2_point function