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Questions:

  1. What is „Nagalase“?
  2. Which role does Nagalase play?
  3. More precisely: „What is „Gc-MAF““?
  4. How is Nagalase thought to inactivate Gc-MAF?
  5. “Nagalase Test”: What does this mean?
  6. I would like a Nagalase test to be performed? How does it work?
  7. What is the cost of the test and how will the test be billed?
  8. Artificially produced Gc MAF for treating diseases
  9. What other parameters can be relevant for a Gc-MAF therapy?
  10. Summary
  11. Scientific references
  12. Explanation of key terms

What is “Nagalase”?

The so-called “Nagalase” is an endogenous enzyme that plays a role in sugar metabolism. The scientifically correct name is “N-acetyl- Galactosaminidase”. Nagalase can split off certain sugar molecules , namely N -acetyl- galactosamine , from large molecules.

Specifically, Nagalase may split off the N- acetyl galactosamine sugar side chain from the so-called “vitamin D -binding protein” (“DBP”).

DBP is found in serum and is also referred to as “Gc-Protein”.

Yamamoto and colleagues reported that tumors can produce Nagalase ( Yamamoto et al. 2008a , 2008b , 2008c , Reddi et al. , 2000). Moreover they reported that in tumor patients Nagalase serum levels mabe increased.

There is evidence that the  Nagalase produced by tumors  has an “immuno- suppressive” effect. It is thought that the Nagalse-mediated immune-suppression in cancer patients contributes to the growth of the existing tumor and the formation of metastases.

The immunosuppressive effect of Nagalase attributed to a mechanism in which Nagalase-mediated removal of sugar moieties  prevents the formation of the immune- stimulating factor Gc -MAF from Gc protein (more information about this mechanism, see below).

It is important to note that in the recent months two publications by Yamamoto and colleagues have been retractred b ythe Editors-In-Chief of the respectiv ejournals. The reasons given for this were “irregularities in the documentation for institutional review board approval“.

However, latest publications by Ward et al. (Am J Immunol, 2014) and Ruggiero et al. (Anticancer Research, 2014) support a possible role of GcMAFin cancer.

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What is the role of Nagalase?

Under normal (“physiologic”) conditions Nagalase is involved in the metabolism of sugar side chains in larger molecules.

The Nagalase produced by tumors is thought to have an immunosuppressive effect ( Saharuddin et al. 2002 , Yamamoto et al. , 1997).

Under diagnostic aspects, Nagalase determination in cancer patients is used to check whether immune-stimulatory therapy is appropriate (“therapy indication”) or to track the response to therapeutic measures (“therapy monitoring”). Particularly Nagalase determination is used when patients are teated with  Gc- MAF.

There is a genetic disease in which the Nagalase synthesis is disturbed: “Schindler’s Disease”. This disease is associated with disorders of the nervous system.

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More precisely: What is “Gc-MAF”?

The Gc-Protein has a sugar side chain consisting of three sugar molecules , namely N – acetyl galactosamine , galactose, and sialic acid  a so-called “tri- saccharide” (= “triple sugar”).

When sialic acid and galactose are removed from this  tri- saccharide, N -acetyl- galactosamine remains as a single sugar moiety.

For Gc-Protein modified in this manner a strong activating effect on macrophages has been described: this modified Gc-Protein is a “Macrophage-Activating Factor”, abbreviated as “MAF”.

More specifically the partially de-glycosylated Gc protein -derived macrophage-activating molecule is called “Gc -MAF” ( Saharuddin et al. 2002 Kuchiike et al. , 2013 Kisker et al. 2003)

It is assumed that this partial sugar elimination (de-glycosylation) from the Gc protein is accomplished under natural conditions by enzymes in immune cells, namely by a so-called “galactosidase” of B-Lymphocytes and a “sialidase” by T-Llymphocytes

Gc -MAF formed by immune cells in inflammatory and defense responses , is thought to have an important role in immune defense, but especially in the immune response against tumors ( Kisker et al. 2003 Thyer et al , 2013B , Uto et al. 2012 Kuchiike et al. , 2013).

I would like to have a Nagalase-Test be performed
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How is Nagalase thought to inactivate Gc-MAF?

The enzyme “Nagalase” completely eliminates the sugar side chain of the Gc protein: the thus-modified Gc protein can no longer be converted to Gc MAF, its immune-stimulatory effect is no longer available.

In other words, the effect of Nagalase on Gc Protein prevents the formation Gc -MAF. Insofar, tumor-derived  Nagalase may have immune-suppressive properties.

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“Nagalase Test”: What does this mean?

There are test procedures, by which the activity of Nagalase can be determined. The Nagalase is isolated from a serum sample and enzyme activity is measured in a quantitative manner.

This test procedure is known as “Nagalase-Test”.

Increased serum Nagalase levels have been described in many diseases, but especially in cancer ( Thyer et al. 2013B , Yamamoto et al. , 1997).

A decline of Nagalase values is interpreted as an as an indication of therapeutic response , especially when monitoring Gc-MAF therapy ( Korbelik et al. 1998 Thyer et al. 2013B , Yamamoto et al. 2008a , 2008b , 2008c ).

I would like to have a Nagalase-Test be performed
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I would like a Nagalase test to be performed? How does it work?

Your doctor may perform the test in collaboration with the medical laboratory of Prof. Kramer in Heidelberg.

For the test, a blood sample (serum) is taken and send in to the laboratory.

Deatailed information on sample generation can be found here.

A request/order form for the test can be found here.

I would like to have a Nagalase-Test be performed
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What is the cost of the test and how will the test be billed?

For privately-insured patients the billing is according to official fees for doctors (GOÄ).

Unfortunately, this test is currently not covered by statutory health insurance and needs to be  be paid by the patient. The price of self-pay is € 67.04.

I would like to have a Nagalase-Test be performed
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Artificially produced Gc-MAF for treating diseases

When the macrophage activating activity  of Gc-MAF became known, research was conducted towards synthetic production of Gc MAF and its use as an immune-stimulating agent.

The therapy with artificial GcMAF is thought to benefit from the fact that artificial GcMAF cannot be inactivated by Nagalase.

Accordingly, methods have been developed for in vitro production of Gc-MAF which is then administered to patients ( Inui et al. , 2013, Thyer et al. 2013A , Uto 2012 , Yamamoto et al. 2008a , 2008b , 2008c ).

The measurement of serum Nagalase activity is used to explore whether artificial Nagalse might be an treatment option (therapy indication) and to assess the effect of Gc-MAf therapy (“therapy – monitoring”).

There are several providers of GcMAF.

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What other parameters may be relevant for Gc -MAF therapy?

Vitamin D levels and serum calcium

The optimum effect of the Gc- MAF appears to depend on optimal vitamin D levels. Recommended values are between 30 and 80 ng / ml.

Therefore, it is recommended to determine the vitamin D value in the serum prior to Gc -MAF therapy and, if necessary, to supplement vitamin D.

In parallel, it is recommended to determine the serum-calcium values and adjust the dose of vitamin D when elevated serum calcium levels are observed.

Genetics of the Vitamin D Receptor ( VDR)

Some authors described (Fabris et al 2012; Pacini et al 2012) that the effect of Gc -MAF depends on the genetic variants of the vitamin D receptor, the so-called “VDR genotypes “. In particular, the VDR variants “Fok 1″ and “BSM1″ were explored.

The best responding patients (“responders”) seem to have the genotype FF / bb, followed by Ff / Bb . The genotype Ff / BB seems to be “low” responders.

Therefore, the information on VDR genotype was taken to set the dose for Gc-MAF treatment.

uPA receptor (uPA -R ) in serum

There are publications that suggest a role of the so-called “uPA receptor” (uPA – R) in the invasive growth of tumors and the formation of metastases. The serum levels of soluble uPA -R have been described as a prognostic factor for tumors ( Soydinç et al. 2012 , Taubert et al. 2010).

In particular a connection between the effect of Gc MAF effect and uPA -R has been described : GC- MAF reduced the amount of uPA -R released by tumor cells ( Gregory et al 2010.).

Some scientists therefore recommend the determination of uPA –R levels in serum as part of a Gc -MAF therapy monitoring in cancer patients.

Genetics of the Gc protein

Genetic variants have also been described for the Gc protein. To what extent these play a role in optimizing Gc- MAF therapy is still the subject of scientific debate.

I would like to have a Nagalase-Test be performed
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Summary

  • Nagalase is an endogenous enzyme in sugar metabolism
  • Tumor cells can produce Nagalase
  • Gc MAF is a macrophage-activating factor , which is produced from Gc-Protein
  • Nagalase produced by tumors can prevet the formation of Gc-MAF from Gc-Protein.
  • Artificially produced Gc-MAF is used as an immune-stimulatory compound
  • The amount of detectable serum Nagalase is measured to check whether a Gc-MAF therapy should be considered (therapy indication) and to monitor the effect of Gc-MAF therapy ( “therapy monitoring”)
  • Additional laboratory parameters are thought to be of importance for Gc -MAF therapy: Vitamin D and calcium levels in blood, the genetic variants of the vitamin D receptor, and the serum levels of soluble uPA receptor.
I would like to have a Nagalase-Test be performed
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Scinetific references

Benis and Schneider

The effects of vitamin D binding protein-macrophage activating factor and colony-stimulation factor-1 on hematopoietic cells in normal and osteopetrotic rats

Blood (1996) 88: 2898 -2905

Fabris et al.

Role of angiotensin – converting enzyme and vitamin D receptor gene polymorphisms in cancer anorexia – cachexia syndrome

Am J Immunol (2012 ) 8: 65-70

Gregory et al.

Vitamin D binding protein – macrophage activating factor directly inhibits proliferation , migration, and uPAR expression of prostate cancer cells

PLoS ONE (2010 ) 5: e13428

Inui et al.

Clinical experience of integrative cancer immunotherapy with GcMAF

Anticancer Res ( 2013) 33: 2917-2920

Kisker et al.

Vitamin D binding protein – macrophage activating factor (DBP – maf ) Inhibits angiogenesis and tumor growth in mice

Neoplasia (2003 ) 5: 32-40

Korbelik et al.

The value of serum alpha -N- acetylgalactosaminidase measurement for the assessment of tumor response to radio- and photodynamic therapy

Br J Cancer (1998) 77 (6): 1009-1014

Kuchiike et al.

Degalactosylated / desialylated human serum Containing GcMAF induced macrophage phagocytic activity and in vivo antitumor activity

Anticancer Res ( 2013) 33: 2881-2886

Nagasawa et al.

Association of the macrophage activating factor ( MAF) precursor activity with polymorphism in vitamin D -binding protein

Anticancer Res ( 2004) 24: 3361-3366

Pacini et al.

Effect of GcMAF on angiogenesis and paracalcitol to the human peripheral blood mononuclear cell proliferation and signaling

J. Nephrol (2012) 25: 577-581

Reddi et al.

Serum alpha -N- acetylgalactosaminidase is associated with diagnosis / prognosis of patients with squamous cell carcinoma of the uterine cervix

Cancer Letters (2000) 158 : 61-64

Ruggiero et al

Oleic acid, deglyvcosylatedvitamin D-binding protein, nitric oxide: a molecular triad made lethal to cancer

Anticancer Res (2014) 34: 3569-3578

Saharuddin et al.

Tumor cell alpha -N- acetylgalactosminidase activity and its involvement in GcMAF -related macrophage activation

Comparative Biochemistry and Physiology Part A (2002) 132 : 1-8

Soydinç et al.

Utitilty of serum and urine uPAR levels for diagnosis of breast cancer

Asian Pacific Journal of Cancer Prevention ( 2012) 13: 2887-2889

Taubert et al.

Co -detection of urokinase plasminogen activator memember of the system in tumour tissue and serum correlates with a poor prognosis for soft- tissue sarcoma patients

Br J Cancer (2010) 102 : 731-737

Thyer et al.

Therapeutic effects of highly purified de – glycosylated GcMAF in the immunotherapy of patients with chronic diseases

Am J Immunol ( 2013A ) 9: 78-84

Thyer et al.

Gc protein -derived macrophage – activating factor alpha -N- acetylgalactosaminidase Decreases levels in advanced cancer patients

OncoImmunology ( 2013B ) 2: e25769

Thyer et al.

A novel role for a major component of the vitamin D axis: vitamin D binding protein -derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages

Nutrients ( 2013C ) 5: 32577-2589

Uto et al.

GcMAF : our next-generation immunotherapy

Nature (2012) 485 : S67 -S70

Wang et al.

Schindler disease : the molecular lesion in the alpha -N- acetylgalactosaminidase gene did infantile neuroaxonal dystrophy Causes of

J Clin Invest ( 1990) 86: 1752-1756

Ward et al.

Clinical experience of cancer immunotherapy integrated with oleicacid complexed with de-glycosylated vitamin D binding protein

Am J Immunol (2014): 23-32

Yamamoto et al.

Prognostic utility of serum alpha -N- acetylgalactosaminidase and immunosuppression resulted from deglycosylation of serum Gc protein in oral cancer patients

Cancer Res ( 1997) 57: 295-299

Yamamoto et al.

Immunotherapy for prostate cancer with Gc protein -derived macrophage – activating factor , GcMAF

Transl Oncol ( 2008a ) 1: 65-72

Yamamoto et al.

Immunotherapy of metastatic colorectal cancer with vitamin D -binding protein -derived macrophage – activating factor , GcMAF

Cancer Immunol Immunother ( 2008b ) 57: 1007-1016

This publication has been retracted by the journal’s Editor-In-Chief “due to irregularities in the documentation for institutional review board approval“

Yamamoto et al.

Immunotherapy of metastatic breast cancer patients with vitamin D -binding protein -derived macrophage activating factor ( GcMAF )

Int J Cancer ( 2008c ) 122 : 461-467

This publication has been retracted by the journal’s Editor-In-Chief “due to irregularities in the documentation for institutional review board approval“

I would like to have a Nagalase-Test be performed
How does it work ? What should I do?

Explanation of key terms

BSM1

BSM1 denotes a genetic variant of the vitamin D receptor. For this genetic variant, three forms are known : bb , Bb and BB

DBP

See: Gc protein

Galactosamine

A chemically defined sugar molecule.

More information at Wikipedia

Galactose

A chemically defined sugar molecule.

More information at Wikipedia

Galactosidase

An enzyme that cleaves at galactose residues.

FOK1

FOK1 refers to a genetic variant of the vitamin D receptor. For this genetic variant, three forms are known : ff , fF and FF

Gc -MAF

also often abbreviated as “GcMAF” is a macrophage – activating factor , which is formed by partial cleavage of the sugar side chain of Gc protein. From the trisaccharide side chain of the Gc protein is galactose and sialic acid, N- acetylgalactosamine remains.

GcMAF

see Gc -MAF

Gc protein

Gc protein is a serum protein which has the ability to bind vitamin D. Thus, it is also known as Vitamin D binding protein (DBP). Gc protein possesses a three-sugar side-chain , these three sugars are a galactose, a sialic acid and a N – actely galaktosmin.

More information at Wikipedia

GOÄ

Official fees for doctors ; applies to the service billing for privately insured patients.

Immunosuppression

“Suppression” means “turning down” and immune suppression refers to the down-regulation of an immune response by a variety of mechanisms , such as diseases, treatment with “immunosuppressive” drugs or environmental influences. “Nagalase” formed by tumors is also thought to have an immunosuppressive effect.

MAF

Abbreviation for “Macrophage Activating Factor”. There are several factors can activate the macrophages; Gc -MAF is one of them.

Macrophages

Macrophages (freely translated from the Greek : “Big Eater” ) are cells of the immune system and play a role in defense reactions of the body , especially in the immune response.

More infomation at Wikipedia

Nagalase

The so-called “Nagalase” is an endogenous enzyme that plays a role in sugar metabolism. The scientifically correct name is “N -Acetyl- Galactosaminidase”. Nagalase can split off from large the sugar molecule N -acetyl- Galactosamine from larger molecules,.

Sialidase

An enzyme that cleaves sialic acid.

Sialic acid

A chemically defined sugar molecule.

More information at Wikipedia

VDR

Stands for “Vitamin D Receptor”. The VDR is a cellular molecule and binds vitamin D. Via binding to VDR the effects of vitamin D are mediated to the cell.

More information at Wikipedia

uPA

Abbreviation for “Urokinase-type Plasminogen Activator”, also known as “urokinase”. This molecule is thought to play  a role in the invasive growth of tumors and the formation of metastases.

More information at Wikipedia

uPA -R

Abbreviation for “uPA receptor”. This molecule on the surface of cells – also tumor cells – binds uPA and is thought to play an  an important role in the invasive growth of tumors and the formation of metastases.

A fraction of the uPA -R produced and released from tumor cells  can be detected as a “soluble uPA -R” in serum.

More information at Wikipedia

I would like to have a Nagalase-Test be performed
How does it work ? What should I do?