Pathology 4400:

Take Home Assignment

Student Name: Seungwon Han

Student Number: XXXXX
Due: March 17, 2023
1. A smoker is looking into quitting cigarettes and is having a hard time and is looking for
some help. They came across varenicline. Explain how varenicline works as a smoking
cessation drug and incorporate dose-response curves into you answer. (6 marks)

Cigarettes contain nicotine, a substance that acts by binding to nicotinic cholinergic

receptors. In mammals, there are as many as nine α subunits (α2 to α10) and three β subunits (β2 to
β4) within the nicotinic cholinergic receptor complex, but the α4β2 receptor is the most common in
the human brain (Benowitz, 2009). Stimulation of nicotinic cholinergic receptors by nicotine
releases dopamine in the brain, along with other neurotransmitters such as norepinephrine,
acetylcholine, serotonin, γ-aminobutyric acid (GABA), and more. Dopamine release in the nucleus
accumbens is associated with drug reward and addiction (Benowitz, 2009).

Whereas nicotine is a full agonist, varenicline is a partial agonist of α4β2 nicotinic

acetylcholine receptors. Varenicline binds with high affinity to α4β2 receptors. Consequently,
varenicline competitively prevents the binding of nicotine to the receptor and blocks the other
effects of nicotine on the receptor (McCarthy & Versella, 2019). Also, as a partial agonist,
varenicline has 50% of the agonist activity of nicotine. Therefore, varenicline still activates the
receptor and helps to alleviate nicotine withdrawal symptoms (McCarthy & Versella, 2019).
Competition of varenicline at increasing doses (as would be taken by someone looking into
quitting cigarettes) against a fixed concentration of nicotine would result in nicotine being
outcompeted (Figure 1).

Figure 1. Competition of full agonist (fixed concentration) and partial agonist (increasing
concentration) for receptor bonding.

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 As seen in Figure 1, as the bonding of agonist to the host receptors decrease, the bonding
of the partial agonist to host receptors increases. The dose response would also look more like that
of varenicline, encouraging the smoker to quit smoking while minimizing withdrawal symptoms
(Figure 2). As seen in Figure 2, the dose response of full agonist decreases and plateaus almost
at zero whereas the dose response of partial agonist increases and plateaus at 0.5. Therefore, the
total response (with elapsed time) ends up looking like the dose response of partial agonist.

Figure 2. Dose response of full agonist (fixed concentration) and partial agonist (increasing
concentration).

2. A friend of yours is a mechanic at an autobody shop and finds a blue Gatorade bottle and
drinks it, unknowing to him it contained windshield washer fluid – which contains
methanol. One of your friends recommend that’s they should drink light beer (contains 4%
ethanol) to minimize any negative effects. Please explain why this would or would not be
helpful. Explain the utility of the osmolar and anion gap in detecting methanol
consumption. (10 marks)

Methanol is toxic. Although methanol is not toxic itself, it is metabolized into

formaldehyde and then formic acid/formate (which is toxic). As little as 15 to 30 mL of 100%
methanol may destroy the optic nerve and cause permanent blindness (Liberski et al., 2022).
Methanol is an alcohol, so when it is first consumed it elevates the osmol gap (which is usually
around 10). Initial symptoms of methanol poisoning are similar to that of ethanol ingestion, like
ataxia (CDC, 2011).

As metabolism occurs in the liver through oxidation with alcohol dehydrogenase (methanol
-> formaldehyde) and aldehyde dehydrogenase (formaldehyde -> formic acid), the osmol gap
decreases but the anion gap increases (Ashurst & Nappe, 2022). This is because formic acid
consumes bicarbonate in the body. Formate accumulates in the body and has many toxic effects.

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For example, it inhibits the enzymes cytochrome oxidase and consequently disrupts oxidative
phosphorylation. Formic acid exerts oxidative stress and causes end organ and retinal damage. It
takes time for methanol metabolism to occur (12 to 24 hours), which is why there is a latency
period after initial ingestion before symptoms of retinal toxicity and metabolic acidosis (Ashurst
& Nappe, 2022).

The osmolar and anion gap should thus be useful in detecting methanol consumption in
conjunction with symptoms and time of ingestion. If the osmol gap is high but the anion gap is
normal and it is within 12 hours of suspected methanol ingestion, there is reason to believe
ingestion of unknown alcohol even without any symptoms. Conversely, if the osmol gap is low
and the anion gap is high and the patient is presenting with blurry vision and hyperventilation and
has consumed the substance in the past >12 hours, methanol ingestion should be suspected. A
definitive diagnosis may be achieved with gas or liquid chromatography (Kraut, 2026).

Figure 3. Changes in osmol and anion gaps with co-ingested ethanol.

The treatment for methanol is venous administration of ethanol or fomepizole, which

inhibits alcohol dehydrogenase from metabolizing methanol to formaldehyde. In addition,
hemodialysis is used to remove methanol and formate in the patient (Kraut, 2016). Ethanol is better
at binding to alcohol dehydrogenase than methanol and thus competitively inhibits methanol from
binding to alcohol dehydrogenase enzyme in the liver. Instead, alcohol dehydrogenase converts
ethanol to the much less toxic acetaldehyde (Pohanka, 2019).

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 Considering how one of the standard treatments is 10% ethanol administered intravenously
(which is not subject to the first pass effect), drinking the light beer (4% ethanol and subject to the
first pass effect) would only be minimally helpful (Ashurst & Nappe, 2022). It may still be able to
keep the osmol gap elevated for longer, thus delaying the formation of the toxic formic
acid/formate metabolite—shown in Figure 3. Ideally, however, the friend should drink stronger
beers (over 10% ethanol, minimum) but also go to the hospital even if he is lured into a false sense
of security during the latency period.

3. A truck driver had a positive urine opioid screen by immunoassay at a routine work place
drug screening service as a mandatory requirement for employment. They claim that they
have recently eaten poppy seed bagels. Explain how the consumption of poppy seed bagels
could cause and positive immunoassay drug screen. Explain how confirmatory/definitive
testing by GC-MS or LC-MSMS may or may not help if the suspected substance is heroin.
Do you expect to be able to make confident conclusion on the detection of heroin? (8
marks)

Poppy seeds do not contain opium (Lachenmeier et al., 2010). Although more than 90% of
the opium content is removed from the seeds during processing, the seeds can become coated by
or absorb opiate extract due to contamination during harvesting (Lachenmeier et al., 2010). Opium
is a milky substance extracted along with poppy seeds, and 12% of it is composed of morphine
(Thevis et al., 2003). Other opium alkaloids such as codeine, thebaine, noscapine, and papaverine
have been found on poppy seeds (Carlin et al., 2020).

In fact, eating a poppy seed bagel may produce an opiate level of approximately 250 ng/ml
in urine three hours after consumption (Selavka, 1991). If the cut off limit for the immunoassay
was lower, the possibility of a false positive on a drug screening service exists, especially since
the screening test used in the first place will be the cheaper and less specific Tier 1 immunoassay
screen (Lachenmeier et al., 2010). Immunoassays are calibrated against a drug class compound,
usually morphine, and this single workplace test could be used to screen for not just morphine but
other opiates like hydrocodone, oxycodone, and more. The catch is that there is often significant
cross-reactivity with immunoassay drug screen; a positive Tier 1 test therefore requires a Tier 2
follow-up to differentiate within the drug class.

Figure 4. Metabolism of heroin in the body.

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 Tier 2 testing by GC-MS or LC-MSMS may help to determine if the suspected substance
is heroin. Heroine is metabolized into the distinct metabolite 6-O-monoacetylmorphine (6-MAM),
which is detected in urine within 24 hours of heroin use (Figure 4Figure 4) (Cone et al., 1991).
Heroine is metabolized very fast, so the detection window of 6-MAM is narrow for urine, but it is
identifiable in hair for months. The small quantity of opium alkaloids within a poppy seed bagel
is unlikely to produce a false positive in a hair analysis because the small amount of opioid in the
bloodstream would not persist in the bloodstream long enough to be incorporated into hair follicles.
A study comparing hair analysis (levels of morphine, 6-MAM, and codeine) results of actual
heroine-using subjects versus subjects who had ingested 150 g of poppy seeds over a 3 week period
found that all 69 heroin users presented with 2 ng /10 mg to 200 ng MAM /10 mg of hair opiates
(Hill et al., 2005). However, only around half of the heroin users had positive urine 6-MAM tests
(Hill et al., 2005). In contrast, the poppy seeds eaters averaged only 0.17 ng/10 mg hair opiates
(Hill et al., 2005). Clearly, there is a significant difference in the levels of heroine metabolites
found in the hair of actual heroin users versus poppy seed eaters. Therefore, I would be able to
make a confident conclusion on the detection of heroine following a Tier 2 GC-MS or LC-MSMS
hair analysis.

4. You are overseeing a drinking water testing service that monitors for trace metal
contamination. A sample is collected from a personal well on a farm that uses chemical
pesticides/insecticides and the results indicate an elevated total arsenic in the drinking
water. How would you consult on the relative toxicity of the arsenic species and what can
you conclude from a total arsenic measurement? Is any further testing needed, if so please
explain? Please explain the experimental principles of the measurements. (8 marks)

Both inorganic and organic arsenic species are found in water, but inorganic arsenic species
tend to dominate in water (Rajakovic & Rajakovic-Ognjanovic, 2018). The inorganic As species
are arsenite As(III) and arsenate As(V), and these are more toxic than organic species such as
monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) (Yogarajah & Tsai, 2015). The
term “total arsenic count” is often misleading, as organic species of arsenic is not much of a
concern (Schwarcz, 2018). Organic arsenicals, such as arsenobetaine, are found in seafood but are
nontoxic because the arsenic atoms in arsenobetaine are tied up and unavailable for bonding with
important proteins in the body (Schwarcz, 2018). On the other hand, inorganic acid is able to bind
to sulfhydryl groups on proteins, impairing protein function and resulting in lung disease and liver
failure amongst other morbidities (EPA, 2014). Even within inorganic As species, As(III) is 60
times more toxic than As(V) (Ratnaike, 2003).

Most arsenic species in drinking water is inorganic, so it is safe to assume that total arsenic
levels in drinking water is a somewhat accurate representation of the inorganic [toxic] arsenic
species which are relevant to human health (Rajakovic & Rajakovic-Ognjanovic, 2018). Methods
for total arsenic detection in water include inductively coupled plasma atomic emission

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spectroscopy (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) (Rajakovic
& Rajakovic-Ognjanovic, 2018).

Figure 5. Diagram of Inductively Coupled Plasma - Atomic Emission Spectrometry (ICP-

AES).

ICP-AES requires no sample pretreatment. As shown in Figure 5, it exploits the nature of

excited electrons to emit energy of a given wavelength when excited by high temperature argon
plasma before returning to the electrons’ ground states. Each element, in this case arsenic, emits
energy at its wavelength. The light is collected by an optical spectrometer, which is then detected
and analyzed (Michalke & Nischwitz, 2013). However, ICP-AES has a lower sensitivity than ICP-
MS, which is used more frequently. ICP-AES is better at monitoring nonmetallic elements, but
this is not the case with arsenic (Michalke & Nischwitz, 2013).

Figure 6. Diagram of Inductively coupled plasma mass spectrometry (ICP-MS).

As seen in Figure 6, ICP-MS atomizes and ionizes the sample, which generates ions. These
ions go through the interface region and enter electrostatic lenses called the ion optics. The lenses
guide the ion beam into the quadrupole mass analyzer. The quadrupole mass analyzer is then able
to select for ions with a specific mass to charge ratio, the ions of which are measured at the detector

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(Wilschefski & Baxter, 2019). ICP-MS can detect different isotopes of the same element, in this
case arsenic.

However, further testing is still required after a total arsenic measurement to detect the
specific arsenic species in the water. This is because (as aforementioned) different arsenical
species have varying toxicities. Ion chromatography-inductively coupled plasma mass
spectrometry (IC-ICP-MS) and high-performance liquid chromatography hydride generation
atomic absorption spectrometry (HPLC–HG-AAS) are some of the methods for arsenic speciation
in water. HPLC-HG-AAS has a detection limit of 0.05–0.8 μg/L for arsenic species in the water.
IC-ICP-MS has a detection limit of 0.01 μg/L for arsenic species in the water (Rajakovic &
Rajakovic-Ognjanovic, 2018).

Figure 7. Mass spectrometry readouts of inorganic and organic arsenic species using IC-
ICP-MS.

In IC-ICP-MS, IC separates ions based on chemical structure, which leads to molecular

sensitivity (Rosenstraus et al., 1998). ICP-MS then detects for ions with a specific mass to charge
ratio. As seen in Figure 7, IC-ICP-MS thus allows for speciation of inorganic and organic arsenic
in a single run (Michalski, 2016).

Figure 8. Diagram of Atomic Absorption Spectrometry (AAS).

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 In HPLC-HG-AAS, HPLC separation of As is achieved using anion exchange columns for
chromatographic separation (Moldovan et al., 1998). The eluents are then passed through a hydride
generator which converts the separated analytes into volatile hydrides. The hydrides are collected
and passed through atomic absorption spectroscopy. In AAS (Figure 8), a hollow cathode lamp
emits the characteristic spectrum of the element to be determined. A lamp unique to arsenic must
be used. Then, flame and nebulizer further atomize the arsenic. After, a monochromator excludes
all wavelengths except for the wavelength of light absorbed by arsenic. Lastly, a detector measures
the intensity of the light. As shown in Figure 9, HPLC-HG-AAS is thus able to achieve speciation
and detection of arsenical species (Vista, 2015).

Figure 9. Chromatogram readouts of three multispecies arsenical solution a, b, c using

HPLC-HG-AAS to determine arsenical species.

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5. Stacey is a happy and energetic 19-year-old student who went to the Caribbean on vacation
for March break. Stacey had been drinking bottled water and pop with ice from the
kitchen’s ice machine while staying at an all-inclusive resort. A few days into her vacation,
Stacey contracted the hepatitis A virus from the contaminated ice cubes and presented to a
hospital with acute decompensated hepatitis A infection. Describe the time course of viral
RNA and antibody responses you would expect to see a hepatitis A infection. Describe the
clinical course you would expect for a patient presenting with acute hepatitis A infection,
and explain the utility of biomarkers AST, ALT, and bilirubin along with their expected
direction of change, why and when it would occur relative to time since infection. (10
marks)

Hepatitis A virus (HAV) is transmitted fecal-orally, so epidemics of it are common. Stacey

was most likely exposed to Hepatitis A when a worker handling the ice contaminated it with fecal
particles containing HAV (Satar et al., 2000). Hepatitis A is the most common cause of acute viral
hepatitis, but it is not associated with chronic hepatitis. The innate immune system is the frontline
of defense against Hepatitis A, which is an RNA picornavirus (McKnight & Lemon, 2018).

Figure 10. Clinical course of HAV infection.

After exposure to HAV, there is an average incubation of 28 days during which the virus
reaches the intestine and subsequently the liver (Stapleton, 1995). Within the liver, HAV is able
to replicate in vitro noncytopathically (not killing the cells), and the virus is released into the
bloodstream (“viremia”). As shown in Figure 10, this happens early in the infection process when
the person is asymptomatic. The virus is also shed in the feces as early as 1 week in, which means
that people may be asymptomatic and unaware that they have the disease but still be able to spread
the virus to other people. Viremia and fecal shedding happen most extensively just before or
shortly after the patient presents with liver function abnormalities but stops at around the same
time antibody mediated humoral immunity is detected (Stapleton, 1995).

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 Although HAV replicates in the liver without directly killing the hepatocytes, it recruits
virus-specific cytotoxic T cells to the location and cause liver injury. There is growing evidence
that the recruitment of natural killer (NK) cells, and non-HAV-specific CD8+ T cells also
contribute to liver injury in HAV infection. Together, this leads to swollen hepatocytes and
hepatocyte necrosis (Wang & Feng, 2021). And by the 28 day mark, the patient starts presenting
with symptoms of liver injury such as dark urine and jaundice. The timeline for symptoms [clinical
illness] is shown in Figure 10 and indicated in teal. By the time the patient presents with these
symptoms, the initial antibody response against HAV which consists of IgM, IgG, and IgA (not
shown in Figure 10) antibodies can be detected. IgG antibodies are the longest lasting, and they
are the body’s main defense against HAV reinfection (Stapleton, 1995).

When the patient starts presenting with symptoms, they will most likely go to a doctor,
who will order blood tests for HAV diagnosis. HAV is diagnosed by detection of IgM antibodies
in the blood, which will be high during the period of clinical illness, as shown in Figure 10 (WHO,
2022). The doctor may also order additional tests to assess liver injury, as 0.5% of HAV cases lead
to fulminant hepatitis (acute liver failure) (WHO, 2022). Common tests are tests for bilirubin,
Aspartate transaminase (AST), and Alanine transaminase (ALT).

The enzyme levels tests of ALT and AST are used to measure liver injury, but ALT is more
specific to liver injury. ALT is an enzyme in the liver which is released into the bloodstream in
response to hepatocellular injury (National Library of Medicine, 2022). AST level is often elevated
not just in response to liver injury but also to cardiac or skeletal muscle injury. Hepatocellular
injury caused by viral hepatitis will elevate AST and ALT disproportionately to elevations in levels
(Cleveland Clinic, 2023). Cholestatic injuries such as bile-duct obstruction will elevate alkaline
phosphatase (ALP) levels disproportionately to AST and ALT (Green & Flamm, 2002). By testing
ALT and AST levels and comparing them to ALP levels, doctors can determine whether or not
there is liver injury and can even distinguish between hepatocellular and cholestatic injury. ALT
and AST levels rise after infection as HAV multiplies in the liver and reach their peak at week 4,
shortly after the onset of clinical symptoms caused by liver damage (Figure 10). The clinical
symptoms are caused by declining liver function, so the coinciding of elevations of ALT and AST
levels with the onset of symptoms makes sense. Hepatitis A usually resolves on its own without
specific treatment in a span of several weeks to months, usually around the 8 week mark (Johns
Hopkins Medicine, 2021). As seen in Figure 10, the clinical resolution of HAV coincides with the
decrease of ALT levels back to baseline. For reference, the normal AST level is ≤40 IU/mL
(Cleveland Clinic, 2023). In the case of acute viral hepatitis ALT levels increase by 5–10 to > 10
at the peak (Giannini et al., 2005).

AST/ALT ratio is also good to determine whether the problem is acute or chronic. If the
hepatic injury is chronic, the liver’s damage will start to affect other organs, which will elevate
AST. An acute liver injury localized to the liver will not elevate AST to the same extent. Normally,
AST/ALT ratio is lesser than 1. However, an AST/ALT ratio equal to one indicates acute viral

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hepatitis whereas an AST/ALT ratio higher than one may be indicative of cirrhosis (Hall & Cash,
2012).

Figure 11. Change of aminotransferase and bilirubin levels in hepatitis. For acute viral
hepatitis, levels of aminotransferase and bilirubin are shown in blue and orange, respectively. For
acute ischemic hepatitis, levels of aminotransferase and bilirubin are shown in green and yellow,
respectively.

Doctors also test bilirubin levels to determine liver disease. Bilirubin is produced by the
normal breakdown of red blood cells or myoglobin and is then delivered to the liver to be
conjugated and excreted into bile. In healthy people, conjugated bilirubin levels are low. Elevated
levels of conjugated serum bilirubin imply hepatic disease. Concentrations of conjugated bilirubin
of 2 mg/dL or greater (20% of total bilirubin) in serum is classified as conjugated
hyperbilirubinemia (Tripathi & Jialal, 2022). Acute liver disease caused by HAV elevates bilirubin
levels proportionally to the severity of the illness. Bilirubin is elevated by 5-10 times normal levels
at its peak. In the case of acute viral hepatitis, peak bilirubin levels (shown in orange) are reached
shortly after peak ALT levels (shown in blue), as seen on Figure 11. Bilirubin levels taper off with
the resolution of HAV and reach baseline levels around the 12 week mark (Giannini et al., 2005).

Although there is no treatment for HAV specifically, doctors recommend avoiding alcohol
and rest. Doctors will also look out for signs of liver failure and cholestatic hepatitis in the case
the HAV does not resolve on its own.

6. Calvin is a 72-year-old who has lived in a century home for the last 30 years where it was
discovered that he had been living with lead plumbing and lead paint. Recently, Calvin has
been showing signs of cognitive decline. His blood smear demonstrated basophilic
stippling coupled with anemia. Routine blood work showed reduced kidney function.

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 Assuming Calvin is experiencing chronic lead toxicity please describe which tests you
would use to measure his kidney function and stage Calvin’s potential kidney disease. (8
marks)

Long-term chronic exposure to lead may result in lead nephropathy and chronic kidney
disease (CKD). Chronic lead nephropathy is irreversible (Rastogi, 2008). It is diagnosed using
history of lead exposure, blood lead level test, and estimated glomerular filtration rate (eGFR).
CKD is diagnosed using eGFR and urine Albumin: Creatinine Ratio (ACR). Since Calvin already
has an established history of lead exposure, I would order three tests: blood lead level test, eGFR
test, and an ACR test.

Whole blood sample is the sample of choice to measure blood lead levels. The blood is
drawn from a vein without any special preparation for the test. For adults, less than 10 micrograms
per deciliter (µg/dL) or 0.48 micromoles per liter (µmol/L) of lead in the blood is ideal (UCSF,
2020). Individuals with high blood lead level (HBLL) are those whose blood lead level > 10 μg/dL
(Nakhaee et al., 2018).

Figure 12. Stages of CKD.

eGFR is a measure of kidney function. eGFR is measured by a blood test to see creatine
levels in blood. Creatinine is a waste product produced by muscles—healthy kidneys excrete
creatinine in urine. In abnormal kidney function, creatinine builds up in blood. Equations such as
the Cockcroft-Gault Equation are then used to calculate GFR, taking into consideration factors
such as gender, age, and body weight. An eGFR of 90mL/min or higher is considered normal
(National Kidney Foundation, 2023). For example, the average eGFR of 20-29 year old’s is

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116mL/min; 30-39 year old’s is 107mL/min; 40-49 year old’s is 99mL/min. Although eGFR is
expected to decrease with age, eGFR can be used to stage CKD, as seen in Figure 12.

Figure 13. Classification of CKD using GFR and ACR categories.

ACR is used to test for kidney damage. It uses a urine test: dipstick, 24 hour collection, or
random urine sample. Healthy kidneys do not let albumin filter into urine from the blood. A urine
albumin-to-creatinine ratio above 30 mg/g is considered higher than normal (NIDKK). ACR and
eGFR together are used to stage CKD under Kidney Disease Improving Global Outcomes
(KDIGO) 2012 guidelines. CKD is defined as abnormality in function such as eGFR<60 mL/min
or albuminuria ≥30 mg per 24 hours) for more than 3 months (Chen et al., 2019). CKD and ACR
can also be used to screen for risk for CKD, as seen in Figure 13. Calvin would be at risk for CKD
since he was exposed to lead for 30 years and lead is a heavy metal initially stored largely in the
kidneys and liver (Nakhaee et al., 2018). Lead exposure also increases blood pressure, which may
lead to kidney damage (Nakhaee et al., 2018). Therefore, Calvin would benefit from eGFR and
ACR tests.

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 Chronic lead nephropathy occurs as a progressive tubulointerstitial nephritis and can be
detected using eGFR tests, but it is often hard to detect in the early stages. By the time chronic
lead nephropathy may be detected using eGFR, kidney damage is irreversible (Rastogi, 2008).
Other biomarkers of lead nephropathy that can detect damage in the earlier stages include tests for
biological markers of tubular damage (urinary enzyme N-acetylglucosaminidase (NAG) test,
Alkaline phosphatase (ALP) test), tests for cytotoxicity markers (brush border tubular antigens
BBA, BB50 and HF5 that leak as a sign of renal toxicity), or biochemical markers such as increased
urinary excretion of thromboxane (TxB2) and decreased excretion of 6-keto-prostaglandin F1α (6-
keto-PGF1α) which are indicative of damage in the renal medulla (Rastogi, 2008). If Calvin’s
eGFR rates look normal, I would follow up with these more specialized tests that detect earlier
signs of kidney damage.

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