Thymidine kinase
Encyclopedia
Thymidine kinase is an enzyme
, a phosphotransferase
(a kinase
): 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, . It can be found in most living cell
s. It is present in two forms in mammalian cells, TK1 and TK2. Certain viruses also have genetic
information for expression of viral thymidine kinases.
Thymidine kinase catalyses the reaction:
where Thd is deoxythymidine
, ATP
is (energy-rich) adenosine 5’-triphosphate, TMP
is deoxythymidine 5’-phosphate and ADP
is adenosine 5’-diphosphate.
Thymidine kinases have a key function in the synthesis of DNA and thereby in cell division
, as they are part of the unique reaction chain to introduce deoxythymidine into the DNA. Deoxythymidine is present in the body fluids as a result of degradation of DNA from food and from dead cells. Thymidine kinase is required for the action of many antiviral drugs. It is used to select hybridoma
cell lines in production of monoclonal antibodies
. In clinical chemistry it is used as a proliferation marker in the diagnosis, control of treatment and follow-up of malignant disease, mainly of hematological malignancies
.
Somewhat later, it was shown that this incorporation was preceded by phosphorylation
, and, around 1960, the enzyme responsible was purified and characterized.
The Prosite pattern recognises only the cellular type of thymidine kinases.
-dependent),
whereas TK2 is located in mitochondria and is cell cycle-independent.
The genes
of the two types were localized in the mid-1970s.
The gene for TK1 was cloned and sequenced.
The corresponding protein has a molecular weight of about 25 kD. Normally, it occurs in tissue as a dimer. It can be activated by ATP. After activation, it has been converted to a tetramer. The recombinant TK1 cannot be activated and converted to a tetramer in this way, showing that the enzyme occurring in cells has been modified after synthesis.
TK1 is synthesized by the cell during the S phase of cell division. After cell division is completed, TK1 is degraded intracelluarly, so that it does not pass to body fluids after normal cell division.
There is a feed-back regulation of the action of thymidine kinase in the cell: thymidine triphosphate (TTP), the product of the further phosphorylation of thymidine, acts as an inhibitor to thymidine kinase. This serves to maintain a balanced amount of TTP available for nucleic acid synthesis, not oversaturating the system. 5'-Aminothymidine, a non-toxic analogue of thymidine, interferes with this regulatory mechanism and thereby increases the cytotoxicity of thymidine analogues used as antineoplastic drugs.
Genes for virus specific thymidine kinases have been identified in Herpes simplex
virus, Varicella zoster virus and Epstein-Barr virus
.
+ ATP --->
+ ADP
Deoxythymidine reacts with ATP to give deoxythymidine monophosphate and ADP.
and further to deoxythymidine triphosphate by the enzyme nucleoside diphosphate kinase. The triphosphate is included in a DNA molecule, a reaction catalysed by a DNA polymerase
and a complementary DNA molecule (or an RNA molecule in the case of reverse transcriptase
, an enzyme present in retrovirus). Deoxythymidine monophosphate is produced by the cell in two different reactions - either by phosphorylation of deoxythymidine as described above or by methylation of deoxyuridine monophosphate
, a product of other metabolic pathways unrelated to thymidine, by the enzyme thymidylate synthethase
. The second route is used by the cell under normal conditions, and it is sufficient to supply deoxythymidine monophosphate for DNA repair. When a cell prepares to divide, a complete new set-up of DNA is required, and the requirement for building blocks, including deoxythymidine triphosphate, increases. Cells prepare for cell division by making some of the enzymes required during the division. They are not normally present in the cells and are downregulated and degraded afterwards. Such enzymes are called salvage enzyme
s. Thymidine kinase 1 is such a salvage enzyme
, whereas thymidine kinase 2 is not cell cycle
-dependent.
In spite of errors in the technique, it is still used to determine the growth rate of malignant cells and to study the activation of lymphocytes in immunology.
s are cells obtained by fusing tumour cells (which can divide infinitely) and immunoglobulin-producing lymphocytes (plasma cells). Hybridomas can be expanded to produce large quantities of immunoglobulins with a given unique specificity (monoclonal antibodies). One problem is to single out the hybridomas from the large excess of unfused cells after the cell fusion. One common way to solve this is to use thymidine kinase negative (TK-) tumour cell lines for the fusion. The thymidine kinase negative cells are obtained by growing the tumour cell line in the presence of thymidine analogues, that kill the thymidine kinase positive (TK+) cells. The negative cells can then be expanded and used for the fusion with TK+ plasma cells. After fusion, the cells are grown in a medium with methotrexate
or aminopterin
that inhibit the enzyme dihydrofolate reductase
thus blocking the de novo synthesis
of thymidine monophosphate. One such medium that is commonly used is HAT medium
, which contains hypoxanthine
, aminopterin
and thymidine
. The unfused cells from the thymidine kinase-deficient cell line die because they have no source of thymidine monophosphate. The lymphocytes eventually die because they are not "immortal." Only the hybridomas that have "immortality" from their cell line ancestor and thymidine kinase from the plasma cell survive. Those that produce the desired antibody are then selected and cultured to produce the monoclonal antibody.
However, instead of focussing on thymidine kinase, the hybridoma cells can be isolated using the same principle as described with respect to another gene the HGPRT, which synthesises IMP necessary for GMP
nucleotide synthesis in the salvage pathway.
that is only present in anticipation of cell division. The enzyme is not set free from cells undergoing normal division where the cells have a special mechanism to degrade the proteins no longer needed after the cell division. In normal subjects, the amount of thymidine kinase in serum or plasma is therefore very low. Tumour cells release enzyme to the circulation, probably in connection with the disruption of dead or dying tumour cells. The thymidine kinase level in serum therefore serves as a measure of malignant proliferation, indirectly as a measure of the aggressivity of the tumour. It is interesting to note that the form of enzyme present in the circulation does not correspond to the protein as encoded by the gene: the gene corresponds to a protein with molecular weight around 25 kD. It is a dimer with a molecular weight of around 50 kD, if activated by ATP a tetramer with molecular weight around 100 kD. The main fraction of the active enzyme in the circulation has a molecular weight of 730 kD and is probably bound in a complex to other proteins.
The most dramatic increases are seen in hematologic malignancies. The main use of thymidine kinase assay now is in Non-Hodgkin lymphoma
. This disease has a wide range of aggressivity, from slow-growing indolent disease that hardly requires treatment to highly-aggressive rapidly-growing forms that should be treated urgently. This is reflected in the values of serum thymidine kinase, that range from close to the normal range for slow-growing tumours to very high levels for rapidly-growing forms.
Also in dogs, lymphomas cause elevations of serum TK levels, indicative of the disease activity and useful for management of the disease.
Similar patterns can be seen in other hematological malignancies
(leukemia, myeloma myelodysplastic syndrome). A very interesting case is the myelodysplastic syndrome
: Some of them rapidly change to acute leukemia, whereas others remain indolent for very long time. Identification of those tending to change to overt leukemia is important for the treatment.
Also solid tumours give increased values of thymidine kinase. Reports on this have been published for prostatic carcinoma, where thymidine kinase has been suggested as a supplement to PSA (Prostate Specific Antigen)
, the tumor marker
now most frequently used in prostate cancer
. Whereas PSA is considered to give an indication of the tumour mass, thymidine kinase indicates the rate of proliferation. There are also reports of the utility of thymidine kinase measurements in serum in small cell lung cancer
, in breast cancer
and in kidney cancer
.
Non-malignant causes for elevation of thymidine kinase in serum are vitamin B12 deficiency, leading to pernicious anemia, viral infections (particularly by virus from the herpes group) and wound healing after trauma and operation.
and against other virus), as the diseased cells replicate much more frequently than normal cells and also against some non-malignant diseases related to too fast cell replication (for instance psoriasis). There are different classes of drugs to control too fast cell division that are directed against thymidine metabolism and thereby involving thymidine kinase:
Chain terminators are thymidine analogues that are included in the growing DNA chain, but modified so that the chain cannot be further elongated. As analogues of thymidine, they are readily phosphorylated to 5'-monophophates. The monophosphate is further phosphorylated to the corresponding triphosphate and incorporated in the growing DNA chain. The analogue has been modified so that it does not have the hydorxyl group in the 3'-position that is required for continued chain growth. In zidovudine
(AZT; ATC
: ) the 3'-hydroxyl group has been replaced by an azido group, in Stavudine
(ATC
: ) it has been removed without replacement. AZT is used as substrate in one of the methods for determination of thymidine kinase in serum. This implies that AZT interferes with this method and may be a limitation: AZT is a standard component of HAART therapy in HIV infection. One common consequence of AIDS is lymphoma and the most important diagnostic application of thymidine kinase determination is for monitoring of lymphoma.
Other thymidine analogues, for instance Idoxuridine
(ATC
: ) act by blocking base pairing during subsequent replication cycles, thereby making the resulting DNA chain defective. This may also be combined with radioactivity to achieve apoptosis of malignant cells.
Some antiviral drugs, such as acyclovir (ATC
: ) and ganciclovir
(ATC
: ) as well as other recently developed nucleoside analogs make use of the specificity for viral thymidine kinase, as opposed to human thymidine kinases. These drugs act as prodrug
s, which in themselves are not toxic, but are converted to toxic drugs by phosphorylation by viral thymidine kinase. Cells infected with the virus therefore produce highly-toxic triphosphates that lead to cell death. Human thymidine kinase, in contrast, with its more narrow specificity, is unable to phosphorylate and activate the prodrug. In this way, only cells infected by the virus are susceptible to the drug. Such drugs are effective only against viruses from the herpes group with their specific thymidine kinase.
After smallpox
was declared eradicated
by WHO in December 1979, vaccination
programs were terminated. A re-emergence of the disease either by accident or as a result of biological warfare
would meet an unprotected population and could result in an epidemic
that could be difficult to control. Mass vaccination would be unethical
, as the only efficient vaccines against smallpox include live vaccinia
virus with severe adverse effects
on rare occasions. As one protective measure, large amounts of vaccine are kept in stock, but an efficient drug against smallpox has high priority. One possible approach would be to use the specificity of the thymidine kinase of poxvirus for the purpose, in a similar way that it is used for drugs against herpesvirus. One difficulty is that the poxvirus thymidine kinase belongs to the same family of thymidine kinases as the human thymidine kinases and thereby is more similar chemically. The structure of poxvirus thymidine kinases has therefore been determined to find potential antiviral drugs. The search has however not yet resulted in a usable antiviral drug against poxviruses.
The herpesvirus thymidine kinase gene has also been used as a “suicide gene” as a safety system in gene therapy
experiments, allowing cells expressing the gene to be killed using ganciclovir
. This is desirable in case the recombinant gene causes a mutation leading to uncontrolled cell growth (insertional mutagenesis). The thymidine kinase produced by these modified cells may diffuse to neighboring cells, rendering them similarly susceptible to ganciclovir, a phenomenon known as the "bystander effect." This approach has been used to treat cancer in animal models, and is advantageous in that the tumor may be killed with as few as 10% of malignant cells expressing the gene.
A similar use of the thymidine kinase makes use of the presence in some tumor cells of substances not present in normal cells (tumor markers). Such tumor markers are, for instance, CEA (carcinoembryonic antigen) and AFP (alpha fetoprotein). The genes for these tumor markers may be used as promoter genes for thymidine kinase. Thymidine kinase can then be activated in cells expressing the tumor marker but not in normal cells, such that treatment with ganciclovir kills only the tumor cells. Such gene therapy-based approaches are still experimental, however, as problems associated with gene transfer have not yet been completely solved.
Incorporation of a thymidine analogue with boron has been suggested and tried in animal models for boron neutron capture therapy
of brain tumours.
A non-radioactive assay method has been developed by the company Dia-Sorin. In this technique 3'-azido-2',3'-deoxythymidine (AZT)is first phosphorylated to AZT 5'-monophosphate (AZTMP) by TK1 in the sample. AZTMP is measured in a immunoassay with anti-AZTMP antibodies and AZTMP-labeled peroxidase. The assay runs in a closed system on the laboratory robot from DiaSorin
Another newly-developed technique uses a thymidine analogue, bromo-deoxyuridine, as substrate to the enzyme. The product of the reaction (in microtiter plates) binds to the bottom of the wells in the plate. There it is detected with ELISA technique: The wells are filled with a solution of a monoclonal antibody to bromo-deoxyuridine. The monoclonal antibody has been bound (conjugated) to alkaline phosphatase
(an enzyme). After the unbound antibody with attached alkaline phosphatase has been washed away, a solution of a substrate to the alkaline phosphatase, 4-nitrophenyl phosphate, is added. The product of the reaction, 4-nitrophenol
, is yellow at alkaline pH and can be measured by photometry
. This assay gives a considerably more sensitive determination. It is commercially available from the company Biovica.
Direct determination of the thymidine kinase protein by immunoassay has also been used. The amounts of thymidine kinase found by this method did not correlate well with the activities and found to have less clinical significance, and the method has been withdrawn from the market.
The TK1 levels in fetal tissues during development are higher than those of the corresponding tissues later.
Certain non-malignant diseases also give rise to dramatic elevation of TK values in cells and tissue: in peripheric lymphocytes during monocytosis and in bone marrow during pernicious anemia.
As TK1 is present in cells during cell division, it is reasonable to assume that the TK activity in malignant tissue should be higher than in corresponding normal tissue. This is also confirmed in most studies. A higher TK activity is found in neoplastic than in normal tissue, in brain tumours, in hematological malignancies, in cancer and polyps in colon, in breast cancer, in lung cancer, in gastric cancers, in ovarian cancer, in mesotheliomas, in melanomas and in thyroid tumours.
In leukemia and in breast cancer therapy that influences the rate of cell proliferation influences the TK values correspondingly.
The technique has also been validated for lung cancer, for colorectal carcinima, for lung cancer and for renal cell carcinoma.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
, a phosphotransferase
Phosphotransferase
Phosphotransferases are a category of enzymes that catalyze phosphorylation reactions. The general form of the reactions they catalyze is: A—P + B ⇔ B—P + A...
(a kinase
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
): 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, . It can be found in most living cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s. It is present in two forms in mammalian cells, TK1 and TK2. Certain viruses also have genetic
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
information for expression of viral thymidine kinases.
Thymidine kinase catalyses the reaction:
- Thd + ATP → TMP + ADP
where Thd is deoxythymidine
Thymidine
Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...
, ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
is (energy-rich) adenosine 5’-triphosphate, TMP
Thymidine monophosphate
Thymidine monophosphate, also known as 5'-thymidylate, thymidylate, or TMP, is a nucleotide that is used as a monomer in DNA. It is an ester of phosphoric acid with the nucleoside thymidine. TMP consists of a phosphate group, the pentose sugar deoxyribose, and the nucleobase thymine...
is deoxythymidine 5’-phosphate and ADP
Adenosine diphosphate
Adenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....
is adenosine 5’-diphosphate.
Thymidine kinases have a key function in the synthesis of DNA and thereby in cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
, as they are part of the unique reaction chain to introduce deoxythymidine into the DNA. Deoxythymidine is present in the body fluids as a result of degradation of DNA from food and from dead cells. Thymidine kinase is required for the action of many antiviral drugs. It is used to select hybridoma
Hybridoma
Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...
cell lines in production of monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
. In clinical chemistry it is used as a proliferation marker in the diagnosis, control of treatment and follow-up of malignant disease, mainly of hematological malignancies
Hematological malignancy
Hematological malignancies are the types of cancer that affect blood, bone marrow, and lymph nodes. As the three are intimately connected through the immune system, a disease affecting one of the three will often affect the others as well: although lymphoma is technically a disease of the lymph...
.
History
The incorporation of thymidine in DNA was demonstrated around 1950.Somewhat later, it was shown that this incorporation was preceded by phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
, and, around 1960, the enzyme responsible was purified and characterized.
Classification
Two different classes of thymidine kinases have been identified and are included in this super family:- one family groups together thymidine kinase from herpesvirusThymidine kinase from herpesvirusThymidine kinase from herpesvirus is a sub-family of thymidine kinases.Its presence in herpesvirus-infected cells is used to activate a range of antivirals against herpes infection, and thus specifically target the therapy towards infected cells only....
as well as cellular thymidylate kinases,
- the second family groups TK from various sources that include, vertebrates, bacteria, the Bacteriophage T4, poxvirusesPoxviridaePoxviruses are viruses that can, as a family, infect both vertebrate and invertebrate animals.Four genera of poxviruses may infect humans: orthopox, parapox, yatapox, molluscipox....
, African swine fever virusAfrican swine fever virusAfrican swine fever virus is the causative agent of African swine fever . ASFV is a large, double-stranded DNA virus which replicates in the cytoplasm of infected cells, and is the only member of the Asfarviridae family. ASFV infects domestic pigs, warthogs and bushpigs, as well as soft ticks ,...
(ASFV) and Fish lymphocystis disease virus (FLDV). The major capsid protein of insect iridescent viruses also belongs to this family.
The Prosite pattern recognises only the cellular type of thymidine kinases.
Biochemistry
Higher organisms have two isoenzymes, that are chemically very different, TK1 and TK2. The former was first found in fetal tissue, the second was found to be more abundant in adult tissue, and initially they were termed fetal and adult thymidine kinase. Soon it was shown that TK1 is present in the cytoplasm only in anticipation of cell division (cell cycleCell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
-dependent),
whereas TK2 is located in mitochondria and is cell cycle-independent.
The genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
of the two types were localized in the mid-1970s.
The gene for TK1 was cloned and sequenced.
The corresponding protein has a molecular weight of about 25 kD. Normally, it occurs in tissue as a dimer. It can be activated by ATP. After activation, it has been converted to a tetramer. The recombinant TK1 cannot be activated and converted to a tetramer in this way, showing that the enzyme occurring in cells has been modified after synthesis.
TK1 is synthesized by the cell during the S phase of cell division. After cell division is completed, TK1 is degraded intracelluarly, so that it does not pass to body fluids after normal cell division.
There is a feed-back regulation of the action of thymidine kinase in the cell: thymidine triphosphate (TTP), the product of the further phosphorylation of thymidine, acts as an inhibitor to thymidine kinase. This serves to maintain a balanced amount of TTP available for nucleic acid synthesis, not oversaturating the system. 5'-Aminothymidine, a non-toxic analogue of thymidine, interferes with this regulatory mechanism and thereby increases the cytotoxicity of thymidine analogues used as antineoplastic drugs.
Genes for virus specific thymidine kinases have been identified in Herpes simplex
Herpes simplex
Herpes simplex is a viral disease caused by both Herpes simplex virus type 1 and type 2 . Infection with the herpes virus is categorized into one of several distinct disorders based on the site of infection. Oral herpes, the visible symptoms of which are colloquially called cold sores or fever...
virus, Varicella zoster virus and Epstein-Barr virus
Epstein-Barr virus
The Epstein–Barr virus , also called human herpesvirus 4 , is a virus of the herpes family and is one of the most common viruses in humans. It is best known as the cause of infectious mononucleosis...
.
+ ATP ---> + ADPDeoxythymidine reacts with ATP to give deoxythymidine monophosphate and ADP.
Physiological context
Deoxythymidine monophosphate, the product of the reaction catalysed by thymidine kinase, is in turn phosphorylated to deoxythymidine diphosphate by the enzyme thymidylate kinaseDTYMK
Thymidylate kinase also known as deoxythymidylate kinase or dTMP kinase is an enzyme that in humans is encoded by the DTYMK gene. and belongs to thymidylate kinase family of proteins.-Further reading:...
and further to deoxythymidine triphosphate by the enzyme nucleoside diphosphate kinase. The triphosphate is included in a DNA molecule, a reaction catalysed by a DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
and a complementary DNA molecule (or an RNA molecule in the case of reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
, an enzyme present in retrovirus). Deoxythymidine monophosphate is produced by the cell in two different reactions - either by phosphorylation of deoxythymidine as described above or by methylation of deoxyuridine monophosphate
Deoxyuridine monophosphate
Deoxyuridine monophosphate is a deoxynucleotide.It is an intermediate in the metabolism of deoxyribonucleotides.-See also:* uridine monophosphate* DCMP deaminase...
, a product of other metabolic pathways unrelated to thymidine, by the enzyme thymidylate synthethase
Thymidylate synthase
Thymidylate synthetase is the enzyme used to generate thymidine monophosphate , which is subsequently phosphorylated to thymidine triphosphate for use in DNA synthesis and repair....
. The second route is used by the cell under normal conditions, and it is sufficient to supply deoxythymidine monophosphate for DNA repair. When a cell prepares to divide, a complete new set-up of DNA is required, and the requirement for building blocks, including deoxythymidine triphosphate, increases. Cells prepare for cell division by making some of the enzymes required during the division. They are not normally present in the cells and are downregulated and degraded afterwards. Such enzymes are called salvage enzyme
Salvage enzyme
Salvage enzymes are enzymes, nucleoside kinases, required during cell division to "salvage" nucleotides, present in body fluids, for the manufacture of DNA.They catalyze the phosphorylation of nucleosides to nucleoside - 5'-phosphates, that are further phosphorylated to triphosphates, that can be...
s. Thymidine kinase 1 is such a salvage enzyme
Salvage enzyme
Salvage enzymes are enzymes, nucleoside kinases, required during cell division to "salvage" nucleotides, present in body fluids, for the manufacture of DNA.They catalyze the phosphorylation of nucleosides to nucleoside - 5'-phosphates, that are further phosphorylated to triphosphates, that can be...
, whereas thymidine kinase 2 is not cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
-dependent.
Identification of dividing cells
The first indirect use of thymidine kinase in biochemical research was the identification of dividing cells by incorporation of radiolabeled thymidine and subsequent measurement of the radioactivity or autoradiography to identify the dividing cells. For this purpose tritiated thymidine is included in the growth medium.In spite of errors in the technique, it is still used to determine the growth rate of malignant cells and to study the activation of lymphocytes in immunology.
PET scan of active tumours
3'-Deoxy-3'-[(18)F]fluorothymidine is a thymidine analogue. Its uptake is regulated by thymidine kinase 1, and it is therefore taken up preferentially by rapidly proliferating tumour tissue. The fluorine isotope 18 is a positron emitter that is used in positron emission tomography (PET). This marker is therefore useful for PET imaging of active tumour proliferation, and compares favourably with the more commonly used marker 2-[(18)F]fluoro-2-deoxy-D-glucose.Selection of hybridomas
HybridomaHybridoma
Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...
s are cells obtained by fusing tumour cells (which can divide infinitely) and immunoglobulin-producing lymphocytes (plasma cells). Hybridomas can be expanded to produce large quantities of immunoglobulins with a given unique specificity (monoclonal antibodies). One problem is to single out the hybridomas from the large excess of unfused cells after the cell fusion. One common way to solve this is to use thymidine kinase negative (TK-) tumour cell lines for the fusion. The thymidine kinase negative cells are obtained by growing the tumour cell line in the presence of thymidine analogues, that kill the thymidine kinase positive (TK+) cells. The negative cells can then be expanded and used for the fusion with TK+ plasma cells. After fusion, the cells are grown in a medium with methotrexate
Methotrexate
Methotrexate , abbreviated MTX and formerly known as amethopterin, is an antimetabolite and antifolate drug. It is used in treatment of cancer, autoimmune diseases, ectopic pregnancy, and for the induction of medical abortions. It acts by inhibiting the metabolism of folic acid. Methotrexate...
or aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...
that inhibit the enzyme dihydrofolate reductase
Dihydrofolate reductase
- Function :Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids...
thus blocking the de novo synthesis
De novo synthesis
De novo synthesis refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation. For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as...
of thymidine monophosphate. One such medium that is commonly used is HAT medium
HAT medium
HAT Medium is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis...
, which contains hypoxanthine
Hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-Hydroxypurine. Hypoxanthine is a necessary additive in certain cell,...
, aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...
and thymidine
Thymidine
Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...
. The unfused cells from the thymidine kinase-deficient cell line die because they have no source of thymidine monophosphate. The lymphocytes eventually die because they are not "immortal." Only the hybridomas that have "immortality" from their cell line ancestor and thymidine kinase from the plasma cell survive. Those that produce the desired antibody are then selected and cultured to produce the monoclonal antibody.
However, instead of focussing on thymidine kinase, the hybridoma cells can be isolated using the same principle as described with respect to another gene the HGPRT, which synthesises IMP necessary for GMP
Guanosine monophosphate
Guanosine monophosphate, also known as 5'-guanidylic acid or guanylic acid and abbreviated GMP, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside guanosine. GMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase...
nucleotide synthesis in the salvage pathway.
Clinical chemistry
Thymidine kinase is a salvage enzymeSalvage enzyme
Salvage enzymes are enzymes, nucleoside kinases, required during cell division to "salvage" nucleotides, present in body fluids, for the manufacture of DNA.They catalyze the phosphorylation of nucleosides to nucleoside - 5'-phosphates, that are further phosphorylated to triphosphates, that can be...
that is only present in anticipation of cell division. The enzyme is not set free from cells undergoing normal division where the cells have a special mechanism to degrade the proteins no longer needed after the cell division. In normal subjects, the amount of thymidine kinase in serum or plasma is therefore very low. Tumour cells release enzyme to the circulation, probably in connection with the disruption of dead or dying tumour cells. The thymidine kinase level in serum therefore serves as a measure of malignant proliferation, indirectly as a measure of the aggressivity of the tumour. It is interesting to note that the form of enzyme present in the circulation does not correspond to the protein as encoded by the gene: the gene corresponds to a protein with molecular weight around 25 kD. It is a dimer with a molecular weight of around 50 kD, if activated by ATP a tetramer with molecular weight around 100 kD. The main fraction of the active enzyme in the circulation has a molecular weight of 730 kD and is probably bound in a complex to other proteins.
The most dramatic increases are seen in hematologic malignancies. The main use of thymidine kinase assay now is in Non-Hodgkin lymphoma
Non-Hodgkin lymphoma
The non-Hodgkin lymphomas are a diverse group of blood cancers that include any kind of lymphoma except Hodgkin's lymphomas. Types of NHL vary significantly in their severity, from indolent to very aggressive....
. This disease has a wide range of aggressivity, from slow-growing indolent disease that hardly requires treatment to highly-aggressive rapidly-growing forms that should be treated urgently. This is reflected in the values of serum thymidine kinase, that range from close to the normal range for slow-growing tumours to very high levels for rapidly-growing forms.
Also in dogs, lymphomas cause elevations of serum TK levels, indicative of the disease activity and useful for management of the disease.
Similar patterns can be seen in other hematological malignancies
Hematological malignancy
Hematological malignancies are the types of cancer that affect blood, bone marrow, and lymph nodes. As the three are intimately connected through the immune system, a disease affecting one of the three will often affect the others as well: although lymphoma is technically a disease of the lymph...
(leukemia, myeloma myelodysplastic syndrome). A very interesting case is the myelodysplastic syndrome
Myelodysplastic syndrome
The myelodysplastic syndromes are a diverse collection of hematological medical conditions that involve ineffective production of the myeloid class of blood cells....
: Some of them rapidly change to acute leukemia, whereas others remain indolent for very long time. Identification of those tending to change to overt leukemia is important for the treatment.
Also solid tumours give increased values of thymidine kinase. Reports on this have been published for prostatic carcinoma, where thymidine kinase has been suggested as a supplement to PSA (Prostate Specific Antigen)
Prostate specific antigen
Prostate-specific antigen also known as gamma-seminoprotein or kallikrein-3 is a glycoprotein that in humans is encoded by the KLK3 gene. KLK3 is a member of the kallikrein-related peptidase family that are secreted by the epithelial cells of the prostate gland...
, the tumor marker
Tumor marker
A tumor marker is a substance found in the blood, urine, or body tissues that can be elevated in cancer, among other tissue types. There are many different tumor markers, each indicative of a particular disease process, and they are used in oncology to help detect the presence of cancer...
now most frequently used in prostate cancer
Prostate cancer
Prostate cancer is a form of cancer that develops in the prostate, a gland in the male reproductive system. Most prostate cancers are slow growing; however, there are cases of aggressive prostate cancers. The cancer cells may metastasize from the prostate to other parts of the body, particularly...
. Whereas PSA is considered to give an indication of the tumour mass, thymidine kinase indicates the rate of proliferation. There are also reports of the utility of thymidine kinase measurements in serum in small cell lung cancer
Lung cancer
Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. If left untreated, this growth can spread beyond the lung in a process called metastasis into nearby tissue and, eventually, into other parts of the body. Most cancers that start in lung, known as primary...
, in breast cancer
Breast cancer
Breast cancer is cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. Cancers originating from ducts are known as ductal carcinomas; those originating from lobules are known as lobular carcinomas...
and in kidney cancer
Kidney cancer
Kidney cancer is a type of cancer that starts in the cells in the kidney.The two most common types of kidney cancer are renal cell carcinoma and urothelial cell carcinoma of the renal pelvis...
.
Non-malignant causes for elevation of thymidine kinase in serum are vitamin B12 deficiency, leading to pernicious anemia, viral infections (particularly by virus from the herpes group) and wound healing after trauma and operation.
Therapeutic
Some drugs are specifically directed against dividing cells. They can be used against tumours and viral diseases (both against retrovirusReverse transcriptase inhibitor
Reverse-transcriptase inhibitors are a class of antiretroviral drug used to treat HIV infection, tumors, and cancer. RTIs inhibit activity of reverse transcriptase, a viral DNA polymerase enzyme that retroviruses need to reproduce.-Mechanism:...
and against other virus), as the diseased cells replicate much more frequently than normal cells and also against some non-malignant diseases related to too fast cell replication (for instance psoriasis). There are different classes of drugs to control too fast cell division that are directed against thymidine metabolism and thereby involving thymidine kinase:
Chain terminators are thymidine analogues that are included in the growing DNA chain, but modified so that the chain cannot be further elongated. As analogues of thymidine, they are readily phosphorylated to 5'-monophophates. The monophosphate is further phosphorylated to the corresponding triphosphate and incorporated in the growing DNA chain. The analogue has been modified so that it does not have the hydorxyl group in the 3'-position that is required for continued chain growth. In zidovudine
Zidovudine
Zidovudine or azidothymidine is a nucleoside analog reverse-transcriptase inhibitor , a type of antiretroviral drug used for the treatment of HIV/AIDS. It is an analog of thymidine....
(AZT; ATC
Anatomical Therapeutic Chemical Classification System
The Anatomical Therapeutic Chemical Classification System is used for the classification of drugs. It is controlled by the WHO Collaborating Centre for Drug Statistics Methodology , and was first published in 1976....
: ) the 3'-hydroxyl group has been replaced by an azido group, in Stavudine
Stavudine
Stavudine is a nucleoside analog reverse transcriptase inhibitor active against HIV.-History:...
(ATC
Anatomical Therapeutic Chemical Classification System
The Anatomical Therapeutic Chemical Classification System is used for the classification of drugs. It is controlled by the WHO Collaborating Centre for Drug Statistics Methodology , and was first published in 1976....
: ) it has been removed without replacement. AZT is used as substrate in one of the methods for determination of thymidine kinase in serum. This implies that AZT interferes with this method and may be a limitation: AZT is a standard component of HAART therapy in HIV infection. One common consequence of AIDS is lymphoma and the most important diagnostic application of thymidine kinase determination is for monitoring of lymphoma.
Other thymidine analogues, for instance Idoxuridine
Idoxuridine
Idoxuridine is an anti-herpesvirus antiviral drug.It is a nucleoside analogue, a modified form of deoxyuridine, similar enough to be incorporated into viral DNA replication, but the iodine atom added to the uracil component blocks base pairing. It is used only topically due to...
(ATC
Anatomical Therapeutic Chemical Classification System
The Anatomical Therapeutic Chemical Classification System is used for the classification of drugs. It is controlled by the WHO Collaborating Centre for Drug Statistics Methodology , and was first published in 1976....
: ) act by blocking base pairing during subsequent replication cycles, thereby making the resulting DNA chain defective. This may also be combined with radioactivity to achieve apoptosis of malignant cells.
Some antiviral drugs, such as acyclovir (ATC
Anatomical Therapeutic Chemical Classification System
The Anatomical Therapeutic Chemical Classification System is used for the classification of drugs. It is controlled by the WHO Collaborating Centre for Drug Statistics Methodology , and was first published in 1976....
: ) and ganciclovir
Ganciclovir
Ganciclovir INN is an antiviral medication used to treat or prevent cytomegalovirus infections.Ganciclovir sodium is marketed under the trade names Cytovene and Cymevene . Ganciclovir for ocular use is marketed under the trade name Vitrasert...
(ATC
Anatomical Therapeutic Chemical Classification System
The Anatomical Therapeutic Chemical Classification System is used for the classification of drugs. It is controlled by the WHO Collaborating Centre for Drug Statistics Methodology , and was first published in 1976....
: ) as well as other recently developed nucleoside analogs make use of the specificity for viral thymidine kinase, as opposed to human thymidine kinases. These drugs act as prodrug
Prodrug
A prodrug is a pharmacological substance administered in an inactive form. Once administered, the prodrug is metabolised in vivo into an active metabolite, a process termed bioactivation. The rationale behind the use of a prodrug is generally for absorption, distribution, metabolism, and...
s, which in themselves are not toxic, but are converted to toxic drugs by phosphorylation by viral thymidine kinase. Cells infected with the virus therefore produce highly-toxic triphosphates that lead to cell death. Human thymidine kinase, in contrast, with its more narrow specificity, is unable to phosphorylate and activate the prodrug. In this way, only cells infected by the virus are susceptible to the drug. Such drugs are effective only against viruses from the herpes group with their specific thymidine kinase.
After smallpox
Smallpox
Smallpox was an infectious disease unique to humans, caused by either of two virus variants, Variola major and Variola minor. The disease is also known by the Latin names Variola or Variola vera, which is a derivative of the Latin varius, meaning "spotted", or varus, meaning "pimple"...
was declared eradicated
Eradication of infectious diseases
Eradication is the reduction of an infectious disease's prevalence in the global host population to zero. It is sometimes confused with elimination, which describes either the reduction of an infectious disease's prevalence in a regional population to zero, or the reduction of the global prevalence...
by WHO in December 1979, vaccination
Vaccination
Vaccination is the administration of antigenic material to stimulate the immune system of an individual to develop adaptive immunity to a disease. Vaccines can prevent or ameliorate the effects of infection by many pathogens...
programs were terminated. A re-emergence of the disease either by accident or as a result of biological warfare
Biological warfare
Biological warfare is the use of biological toxins or infectious agents such as bacteria, viruses, and fungi with intent to kill or incapacitate humans, animals or plants as an act of war...
would meet an unprotected population and could result in an epidemic
Epidemic
In epidemiology, an epidemic , occurs when new cases of a certain disease, in a given human population, and during a given period, substantially exceed what is expected based on recent experience...
that could be difficult to control. Mass vaccination would be unethical
Ethics
Ethics, also known as moral philosophy, is a branch of philosophy that addresses questions about morality—that is, concepts such as good and evil, right and wrong, virtue and vice, justice and crime, etc.Major branches of ethics include:...
, as the only efficient vaccines against smallpox include live vaccinia
Vaccinia
Vaccinia virus is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, and which encodes for approximately 250 genes. The dimensions of the virion are roughly 360 × 270 × 250 nm, with a mass of...
virus with severe adverse effects
Adverse effect (medicine)
In medicine, an adverse effect is a harmful and undesired effect resulting from a medication or other intervention such as surgery.An adverse effect may be termed a "side effect", when judged to be secondary to a main or therapeutic effect. If it results from an unsuitable or incorrect dosage or...
on rare occasions. As one protective measure, large amounts of vaccine are kept in stock, but an efficient drug against smallpox has high priority. One possible approach would be to use the specificity of the thymidine kinase of poxvirus for the purpose, in a similar way that it is used for drugs against herpesvirus. One difficulty is that the poxvirus thymidine kinase belongs to the same family of thymidine kinases as the human thymidine kinases and thereby is more similar chemically. The structure of poxvirus thymidine kinases has therefore been determined to find potential antiviral drugs. The search has however not yet resulted in a usable antiviral drug against poxviruses.
The herpesvirus thymidine kinase gene has also been used as a “suicide gene” as a safety system in gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
experiments, allowing cells expressing the gene to be killed using ganciclovir
Ganciclovir
Ganciclovir INN is an antiviral medication used to treat or prevent cytomegalovirus infections.Ganciclovir sodium is marketed under the trade names Cytovene and Cymevene . Ganciclovir for ocular use is marketed under the trade name Vitrasert...
. This is desirable in case the recombinant gene causes a mutation leading to uncontrolled cell growth (insertional mutagenesis). The thymidine kinase produced by these modified cells may diffuse to neighboring cells, rendering them similarly susceptible to ganciclovir, a phenomenon known as the "bystander effect." This approach has been used to treat cancer in animal models, and is advantageous in that the tumor may be killed with as few as 10% of malignant cells expressing the gene.
A similar use of the thymidine kinase makes use of the presence in some tumor cells of substances not present in normal cells (tumor markers). Such tumor markers are, for instance, CEA (carcinoembryonic antigen) and AFP (alpha fetoprotein). The genes for these tumor markers may be used as promoter genes for thymidine kinase. Thymidine kinase can then be activated in cells expressing the tumor marker but not in normal cells, such that treatment with ganciclovir kills only the tumor cells. Such gene therapy-based approaches are still experimental, however, as problems associated with gene transfer have not yet been completely solved.
Incorporation of a thymidine analogue with boron has been suggested and tried in animal models for boron neutron capture therapy
Boron Neutron Capture Therapy
Boron neutron capture therapy is an experimental form of radiotherapy that uses a neutron beam that interacts with boron injected into a patient...
of brain tumours.
In serum
The level of thymidine kinase in serum or plasma is so low that the measurement is best based on the enzymatic activity. In commercial assays, this is done by incubation of a serum sample with a substrate analogue. The oldest commercially-available technique uses iodo-deoxyuridine wherein a methyl group in thymidine has been replaced with radioactive iodine. This substrate is well accepted by the enzyme. The monophosphate of iododeoxyuridine is adsorbed on aluminium oxide that is suspended in the incubation medium. After decantation and washing the radioactivity of the aluminium oxide gives a measure of the amount of thymidine kinase in the sample. Kits using this principle are commercially available from the companies Immunotech/Beckman and DiaSorin.A non-radioactive assay method has been developed by the company Dia-Sorin. In this technique 3'-azido-2',3'-deoxythymidine (AZT)is first phosphorylated to AZT 5'-monophosphate (AZTMP) by TK1 in the sample. AZTMP is measured in a immunoassay with anti-AZTMP antibodies and AZTMP-labeled peroxidase. The assay runs in a closed system on the laboratory robot from DiaSorin
Another newly-developed technique uses a thymidine analogue, bromo-deoxyuridine, as substrate to the enzyme. The product of the reaction (in microtiter plates) binds to the bottom of the wells in the plate. There it is detected with ELISA technique: The wells are filled with a solution of a monoclonal antibody to bromo-deoxyuridine. The monoclonal antibody has been bound (conjugated) to alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
(an enzyme). After the unbound antibody with attached alkaline phosphatase has been washed away, a solution of a substrate to the alkaline phosphatase, 4-nitrophenyl phosphate, is added. The product of the reaction, 4-nitrophenol
4-Nitrophenol
4-Nitrophenol is a phenolic compound that has a nitro group at the opposite position of hydroxy group on the benzene ring.-Properties:...
, is yellow at alkaline pH and can be measured by photometry
Photometer
In its widest sense, a photometer is an instrument for measuring light intensity or optical properties of solutions or surfaces. Photometers are used to measure:*Illuminance*Irradiance*Light absorption*Scattering of light*Reflection of light*Fluorescence...
. This assay gives a considerably more sensitive determination. It is commercially available from the company Biovica.
Direct determination of the thymidine kinase protein by immunoassay has also been used. The amounts of thymidine kinase found by this method did not correlate well with the activities and found to have less clinical significance, and the method has been withdrawn from the market.
In tissue
Thymidine kinase has been determined in tissue samples after extraction of the tissue. No standard method for the extraction or for the assay has been developed and TK determination in extracts from cells and tissues have not been validated in relation to any specific clinical question, see however Romain et al. and Arnér et al. A method has been developed for specific determination of TK2 in cell extracts using the substrate analogue 5-Bromovinyl 2'-deoxyuridine. In the studies referred to below the methods used and the way the results are reported are so different that comparisons between different studies are not possible.The TK1 levels in fetal tissues during development are higher than those of the corresponding tissues later.
Certain non-malignant diseases also give rise to dramatic elevation of TK values in cells and tissue: in peripheric lymphocytes during monocytosis and in bone marrow during pernicious anemia.
As TK1 is present in cells during cell division, it is reasonable to assume that the TK activity in malignant tissue should be higher than in corresponding normal tissue. This is also confirmed in most studies. A higher TK activity is found in neoplastic than in normal tissue, in brain tumours, in hematological malignancies, in cancer and polyps in colon, in breast cancer, in lung cancer, in gastric cancers, in ovarian cancer, in mesotheliomas, in melanomas and in thyroid tumours.
In leukemia and in breast cancer therapy that influences the rate of cell proliferation influences the TK values correspondingly.
Immunohistochemical staining for thymidine kinase
Antibodies against thymidine kinase are available for immunohistochemical detection. Staining for thymidine kinase was a reliable technique for identification of patients with stage 2 breast carcinoma. The highest number of patients identified was obtained by combination of thymidine kinase and Ki-67 staining.The technique has also been validated for lung cancer, for colorectal carcinima, for lung cancer and for renal cell carcinoma.
See also
- Thymidine kinase 1Thymidine kinase 1Thymidine kinase 1, soluble , is a human thymidine kinase.Two forms of this protein have been identified in animal cells, one in cytosol and one in mitochondria...
- Thymidine kinase from herpesvirusThymidine kinase from herpesvirusThymidine kinase from herpesvirus is a sub-family of thymidine kinases.Its presence in herpesvirus-infected cells is used to activate a range of antivirals against herpes infection, and thus specifically target the therapy towards infected cells only....
- Thymidylate kinaseThymidylate kinaseThymidylate kinase catalyzes the phosphorylation of thymidine 5'-monophosphate to form thymidine 5'-diphosphate in the presence of ATP and magnesium:...
- Nucleoside-diphosphate kinaseNucleoside-diphosphate kinaseNucleoside-diphosphate kinases are enzymes that catalyze the exchange of phosphate groups between different nucleoside diphosphates...
- Thymidylate synthaseThymidylate synthaseThymidylate synthetase is the enzyme used to generate thymidine monophosphate , which is subsequently phosphorylated to thymidine triphosphate for use in DNA synthesis and repair....
Further reading
Three survey articles on different aspcets of thymidine kinase are available from the internet site of Biovica International:- Gronowitz JS, Fischer LN, Källander CFR (1996): Thymidine kinase: Biochemical Background and Clinical Applications. I. Background and Use in Gene Therapy. http://biovica.com/wp-content/uploads/2011/05/Thymidine_Gene_therapy.pdf
- Gronowitz JS, Fischer LN, Källander CFR (1996): Thymidine kinase: Biochemical Background and Clinical Applications. II. The quantitative assay for TK and its clinical use. http://biovica.com/wp-content/uploads/2011/05/Thymidine_Quantitative_assay.pdf
- Gronowitz JS, Fischer LN, Källander CFR (1996): Thymidine kinase: Biochemical Background and Clinical Applications. III. Use of TK assay in serum, plasma and other body fluids. http://biovica.com/wp-content/uploads/2011/05/Thymidine_Serum_plasma.pdf