Nitrostress test – nitrosative stress in urine

4-hydroxynitrophenylacetic acid (also 3-nitro-4-hydroxyphenylacetic acid) forms when reactive nitrogen compounds such as peroxynitrite attach a nitro group to tyrosine structures. It is a breakdown product of free nitrotyrosine and is excreted in urine. An elevated value therefore shows that more nitration is taking place in the body.

However, the acid can also form through nitration of 4-hydroxyphenylacetic acid, a breakdown product of the amino acid tyrosine that gut bacteria also produce. The value therefore does not depend on nitrotyrosine alone and is considered together with the other values. Sources: PubMed 12797864, PubMed 25825822.

Methylmalonic acid is broken down in the mitochondria by an enzyme that requires vitamin B12. If the cells lack effective B12, it accumulates and more of it is excreted in urine. An elevated value is therefore a well-studied indication of vitamin B12 deficiency at the cellular level, even when the B12 level in the blood still looks normal.

Elevated values also occur with reduced kidney function and with rare inherited metabolic disorders. Sources: PubMed 32707915, PubMed 12002408. More on the methylmalonic acid page.

Vitamin B12 deficiency can show up in the following ways, among others (PubMed 23301732):

• Tiredness and reduced performance
• Anaemia with pallor and shortness of breath on exertion
• Tingling, numbness or burning in the hands and feet
• Unsteady gait
• Problems with concentration and memory, mood changes
• Burning tongue or a smooth, reddened tongue

Because nerve damage from B12 deficiency can be permanent, such complaints should be checked by a doctor promptly.

Citrulline forms, among other things, when the body produces nitric oxide (NO) from arginine. Environmental medicine therefore uses the ratio of citrulline to creatinine in urine as an indication of increased NO production. How meaningful the value is for this has not been conclusively studied.

Citrulline also has other sources: it is produced mainly in the lining of the small intestine, forms in the urea cycle and occurs in foods such as watermelon and in citrulline or arginine supplements. More on the citrulline page.

Which complaints research associates with nitrosative stress is described under "Which complaints and symptoms is nitrosative stress associated with?".

The body makes the messenger nitric oxide (NO) from the amino acid arginine. NO is needed, for example, for the width of blood vessels and for the immune system. When NO meets oxygen radicals (superoxide), peroxynitrite forms – a highly reactive nitrogen compound.

Nitrosative stress describes reactive nitrogen compounds like this chemically altering proteins, fats or DNA. A well-known example is the formation of nitrotyrosine. The term is closely related to oxidative stress, which comes from reactive oxygen compounds.

What does this mean for the body? Small amounts of reactive nitrogen compounds are normal and are neutralised by the body's own protective systems. If more is produced over time than these systems can handle, proteins, enzymes and energy production in the mitochondria can be impaired (PubMed 18395525, PubMed 11223641). This state is what the term "nitrostress" refers to.

What the tested values show:

• 4-hydroxynitrophenylacetic acid: a trace of the nitration of protein building blocks (nitrotyrosine pathway)
• Citrulline: formed, among other things, when the body produces nitric oxide from arginine
• Methylmalonic acid: shows whether the vitamin B12-dependent energy metabolism in the mitochondria is running smoothly; elevated mainly in vitamin B12 deficiency

What it is associated with: Research associates nitrosative stress with migraine, persistent exhaustion and ME/CFS, and chronic inflammation; vitamin B12 deficiency with tiredness, anaemia, tingling or numbness in the hands and feet, and problems concentrating. How well each link is supported is described under "Which complaints and symptoms …" and "Elevated methylmalonic acid in urine". Such complaints have many possible causes and should first be checked by a doctor.

Biochemically, two things are needed: a lot of nitric oxide and, at the same time, a lot of oxygen radicals. Factors discussed in the scientific literature include inflammatory processes, infections, environmental exposures and sustained physical or psychological strain.

Laboratory values alone cannot show what role such factors play in an individual person.

Not directly: peroxynitrite breaks down within fractions of a second. What is measured instead are metabolites formed in these processes and excreted in urine.

This urine test measures three such values in second morning urine and relates them to creatinine. That makes samples comparable even though urine is diluted differently depending on how much you drink.

Mitochondria are where cells generate energy (ATP) from nutrients. Biochemical studies have described that reactive nitrogen compounds can impair mitochondrial enzymes.

Methylmalonic acid is broken down in the mitochondria by an enzyme that needs vitamin B12 as a helper. That is why laboratory medicine also uses methylmalonic acid as a marker of vitamin B12 status. An elevated value can therefore have various reasons; interpreting it belongs in the results consultation.

Nitric oxide (NO) is a well-studied messenger in migraine research. Glyceryl trinitrate, a substance that releases NO in the body, triggers an attack after a few hours in many people with migraine; this is an established research model. Smaller studies have also observed elevated markers of nitrosative stress in people with migraine. Sources: PubMed 37569369, PubMed 38902612, PubMed 17202769.

Whether the urine values from this test say anything about an individual person's migraine has not been studied. Headaches that are new, change or come with paralysis, speech or vision problems need to be checked by a doctor immediately.

Nitrosative stress is not a distinct condition in medical guidelines, and measuring it is not part of routine diagnostics at a GP practice. It is offered mainly in environmental and preventive medicine.

Laboratories do not assess the urine values uniformly. Your report therefore always states the reference range of the laboratory that carried out the analysis.

Nitrosative stress is not a condition in its own right but a biochemical state. Research associates it with the following complaints and conditions, with varying strength of evidence:

• Migraine: nitric oxide triggers migraine attacks in the research model; smaller studies have found elevated markers of nitrosative stress (PubMed 38902612, PubMed 17202769).
• Persistent exhaustion and ME/CFS: discussed as a possible contributing cause, so far mainly in hypotheses and reviews (PubMed 19127706).
• Chronic inflammation: during inflammation, immune cells produce more nitric oxide; protein nitration is well described there (PubMed 18395525).
• Difficulty concentrating and reduced resilience: often mentioned in environmental-medicine practice, but hardly studied scientifically.

For elevated methylmalonic acid there is also a well-established link with vitamin B12 deficiency and its symptoms – see "Elevated methylmalonic acid in urine".

All of these complaints have many possible causes, from anaemia and thyroid problems to sleep disorders or psychological strain. They should first be checked by a doctor. A urine test cannot explain complaints and does not provide a diagnosis.

If exhaustion is new, increases quickly or comes with fever, night sweats, unintended weight loss, shortness of breath, chest pain or persistent low mood, the first step is a doctor's practice, not a self-test. Common causes such as anaemia, thyroid or metabolic disorders can be checked there with simple examinations.

If you are thinking about harming yourself, get help immediately – in Germany, for example, from TelefonSeelsorge on 0800 111 0 111 (free, 24/7) or emergency number 112.

This page does not give treatment recommendations. Whether a result means anything at all and which steps make sense depends on your overall situation: complaints, previous findings, medication and circumstances.

That belongs in a conversation with the ORY expert team or with your doctor or naturopath, for example from the ORY expert network. The results consultation is included in the price.

Analysis usually takes 7–21 days from the arrival of the sample at the laboratory. As soon as your report is ready, we let you know.

The report lists every value with its unit and the laboratory's reference range. A results consultation with the expert team is included in the price.

The Nitrostress test costs €100 including VAT, shipping, return postage and results consultation.

Statutory health insurance in Germany generally does not cover tests you order yourself. With private insurance, reimbursement may be possible; how it works is described above under "Reimbursement by private insurance".

The cell stress profile measures the same values plus 8-OHdG (8-hydroxydeoxyguanosine), a marker of oxidative changes to DNA. If you want to look at nitrosative and oxidative stress in one sample, you will find both there.

Which scope makes sense for you can be clarified in the free initial consultation.