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Nitrate/Nitrite Toxicity
Clinical Assessment - Laboratory Tests

Course: WB 2342
CE Original Date: December 5, 2013
CE Renewal Date: December 5, 2015
CE Expiration Date: December 5, 2017
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Learning Objectives

Upon completion of this section, you will be able to

  • Identify the laboratory test results that indicate methemoglobinemia.


The evaluation of cyanosis in an infant should systematically work through the differential diagnoses with special emphasis on airway, pulmonary and circulatory causes. In cases of severe cyanosis, urgent supportive therapy (i.e. intravenous fluids, "thermoneutral" environment, glucose infusions/monitoring, airway or assisted ventilation depending on clinical presentation of patient/level of respiratory distress, etc.) should be provided while a diagnosis is established [Steinhorn 2008].

A typical cyanosis workup includes CBC with differential and peripheral blood smear, free serum hemoglobin and haptoglobin, ABGs and pulse oximetry.

Imaging (chest x-ray) and/or functional studies (echocardiogram, EKG) to assess cardiac and/or pulmonary status may be necessary based on clinical presentation.

Increased suspicion for methemoglobinemia is central to timely and accurate diagnosis. Methemoglobin results in distinct changes in blood color and oxygen carrying capacity.

Methemoglobinemia can be acquired (exposure to oxidants) or inherited (i.e. decreased enzyme activity or presence of hemoglobin M). Acquired methemoglobinemia will have normal enzyme assay activity tests and normal Hb electrophoresis. For hereditary methemoglobinemias, reduced enzyme activity is seen with NADH-methemoglobin reductase deficiency, but normal in HbM disease. Hemoglobin electrophoresis is abnormal in HbM disease, but normal with NADH-methemoglobin reductase deficiency [McKenzie 2010].

Table 4 summarizes suggested lab tests for a methemoglobinemia work up.

Screening Tests Included in a Methemoglobinemia work up

A typical methemoglobin work up includes [Denshaw-Burke 2013].

Tests to rule out hemolysis include CBC with differential, reticulocyte count, peripheral blood smear, lactate dehydrogenase (LDH), bilirubin, serum haptoglobin, free serum hemoglobin and Heinz body preparation.

The CBC with differential, the RDW, erythrocyte indices and peripheral blood smear can help identify and characterize anemias, distinguish thalassemias from hemoglobinopathies and detect other abnormalities related to the differential diagnoses for cyanosis.

Free serum hemoglobin and haptoglobin levels are drawn to assess for hemolytic anemias. A decrease in haptoglobin can support a diagnosis of hemolytic anemia when seen with an increased reticulocyte count, decreased erythrocyte count, decreased hemoglobin and hematocrit.

Tests to determine end-organ dysfunction or failure may include liver function tests, electrolytes, renal function tests.

Tests to determine functional or structural abnormalities may include imaging studies of the chest, EKG and echocardiogram.

Testing should include a urine pregnancy test for females of childbearing age to guide treatment and management decisions.

Tests to determine oxygen saturation may include (depending on availability)

ABG and standard pulse oximetry (a "saturation gap" or difference between the oxygen saturation results of ABG alone (calculated) vs. standard pulse oximetry will be present in methemoglobinemia), ABG with co-oximetry, or multiple wavelength pulse oximetry (also called continuous pulse co-oximetry).

Bedside Testing for MetHb

A screening test for methemoglobinemia that can be done at the bedside is described below:

  • Place 1 or 2 drops of the patient's blood on white filter paper.
  • The chocolate-brown appearance of methemoglobin (MetHb) does not change with time.
  • In contrast, deoxyhemoglobin appears dark red/violet initially and then brightens after exposure to atmospheric oxygen.
  • Gently blowing supplemental oxygen onto the filter paper hastens the reaction with deoxyhemoglobin, but does not affect MetHb [Wright et al. 1999; Wentworth et al. 1999; Haymond et al. 2005; Skold et al. 2011; Denshaw-Burke 2013].
  • A tube of MetHb-containing blood will not turn red when shaken in air or when oxygen is bubbled through it, whereas blood that is dark because of normal deoxyhemoglobin will turn red [Henretig et al. 1988; Haymond et al. 2005; Skold et al. 2011; Ritchey et al. 2012; Denshaw-Burke 2013].

Standard Pulse-oximetry and Arterial Blood Gases

Standard Pulse-oximetry measurement of the oxygen saturation of hemoglobin does not provide accurate results in the presence of methemoglobinemia [Ralston et al. 1991; Flomenbaum et al. 2006; DeBaun et al. 2011; Skold et al. 2011; Denshaw-Burke 2013].

  • Standard pulse oximetry underestimates oxygen saturation at low levels of methemoglobinemia and overestimates oxygen saturation when methemoglobinemia is severe (i.e. lower and higher MetHb levels will show a constant oxygen saturation close to 85%).
  • Arterial blood gas analysis will typically reveal a normal arterial oxygen tension (PO2) and may reveal a metabolic acidosis proportional to the severity and duration of tissue hypoxia.
  • ABGs indicate plasma oxygen content and therefore don't correspond to the oxygen-carrying capacity of hemoglobin.
  • The profound and disproportionate metabolic acidosis seen in young infants with diarrheal illness and methemoglobinemia suggests that the acidosis is a cause or coexisting finding rather than a result of methemoglobinemia [Bradberry 2003; Avner et al. 1990; Nelson and Hostetler 2003; DeBaun 2011].

Co-Oximetry and MetHb Levels

MetHb percentages can only be used to estimate oxygen-carrying capacity when interpreted with the total hemoglobin [Osterhoudt 2001; Skold et al. 2011; DeBaun et al. 2011; Denshaw-Burke 2013].

  • Many hospital laboratories do not measure oxygen saturation directly on blood gas analysis. Instead, they derive it from a nomogram that is based on the measured PO2 and the presence of normal hemoglobin. In this case, since standard pulse oximetry assumes and is limited to the absorbance characteristics for oxy and deoxyhemoglobin, the calculated oxygen saturation could be falsely elevated in the presence of methemoglobinemia (depending on amount of MetHb present; doesn't distinguish between the overlapping absorbance characteristics of MetHb).
  • A "saturation gap" exists when the measured oxygen saturation of blood differs from the oxygen saturation calculated by routine blood gas analysis.
  • A saturation gap of more than 5% suggests the presence of MetHb, carboxyhemoglobin, or sulfhemoglobin [Coleman and Coleman 1996; Park and Nagel 1984; Skold et al. 2011; Flomenbaum et al. 2006; DeBaun et al. 2011].

Co-oximetry is an accurate method of measuring MetHb [Skold et al. 2011; Denshaw-Burke 2013].

  • A co-oximeter is a simplified spectrophotometer, but unlike a standard pulse oximeter that only measures absorbance at two wavelengths, it measures light absorbance at multiple different wavelengths to accurately measure the total amount of hemoglobin.
  • These wavelengths correspond to specific absorbance characteristics including
    • Deoxyhemoglobin (reduced hemoglobin),
    • Oxyhemoglobin,
    • Carboxyhemoglobin,
    • Methemoglobin, and
    • Hemoglobin.
  • Interpreting the results from a blood gas analyzer without co-oximetry may lead to misdiagnosis because the oxygen saturation will have been calculated but not measured [Matthews 1995; Mansouri and Lurie 1993; Skold et al. 2011].
  • Sulfhemoglobin and methemoglobin have similar wavelengths which should be considered when interpreting co-oximetry readings. A "pseudomethemoglobinemia" occurs when sulfhemoglobin is erroneously detected as methemoglobin which results in a falsely elevated MetHb level.
  • Note that lipemic blood specimens may also result in a falsely elevated methemoglobin level.

Pulse Co-Oximetry

Multiple wavelength pulse "co-oximeters" now exist and provide a noninvasive and continuous way to measure MetHb levels and oxygen saturation. Some models can distinguish sulfhemoglobin from methemoglobin [Steinhorn 2008; Macknet et al. 2007; Macknet et al. 2010].

Cyanide Test

This test can both quantify MetHb level and distinguish between sulfhemoglobin and MetHb. Cyanide binds to the positively charged MetHb. This binding eliminates the MetHb light absorption wavelengths in direct proportion to the MetHb concentration. MetHb is given as a percentage of total concentration of hemoglobin [Evelyn and Malloy 1938; Skold et al. 2011].

  • Methemoglobin reacts with cyanide to form cyanomethemoglobin which has a bright red color. Sulfhemoglobin doesn't react with cyanide to create this bright red color [Evelyn and Malloy 1938; Skold et al. 2011; Denshaw-Burke 2013].
  • Since methemoglobin has increased red blood cell affinity for cyanide, it can be used in the treatment of cyanide poisoning. Nitrites can be used to oxidize hemoglobin to methemoglobin which can then bind cyanide.

Suggested Laboratory Tests

Table 4. Suggested Lab Tests for Methemoglobinemia Work Up

Test Purpose
Screening Tests
  • Examination of blood color (will stay brown in presence of oxygen)
  • Determination of MetHb level
  • Determination of the calculated versus measured arterial saturation gap using co-oximetry
  • Tests to rule out hemolysis and/or characterize any identified anemia (Complete blood count (CBC) with differential,
  • reticulocyte count, peripheral blood smear including Heinz body preparation, lactate dehydrogenase (LDH), bilirubin, serum free hemoglobin and serum haptoglobin.
  • Tests to determine end-organ dysfunction or failure (liver function tests, electrolytes, renal function tests)
  • Urine pregnancy tests for females of childbearing age (to guide treatment and management decisions)
  • Urinalysis (for hemolysis detection- reddish brown color)
  • If available, ABG with co-oximetry or multiple wavelength pulse oximetry
  • Potassium cyanide test ( can distinguish methemoglobin from sulfhemoglobin)
  • Diagnostic imaging to exclude pulmonary or cardiac disease/abnormalities
    • Imaging studies of chest
    • Echocardiography
    • EKG
Specialized Tests to assess congenital Methemoglobinemia

Tests for causes of congenital methemoglobinemia (i.e. deficiencies of MetHb reducing enzymes or hemoglobin M) include

  • Hemoglobin electrophoresis and DNA sequencing of globin chain gene (to detect hemoglobin M (HbM) variants which have mutations in the globin chain that stabilize heme iron in the ferric state and may give rise to misleading co-oximetry results)
  • Specific enzyme assays (often in several cell lines)
    • Activity of NADH dependent MetHb reductase (also called cytochrome b5 reductase)
    • Activity of glucose 6 phosphate dehydrogenase (G6PD)
    • Activity of NADPH dependent MetHb reductase

Direct Biologic Indicators

In general, measurements of nitrates or nitrites in blood, urine, or saliva are not clinically useful.

Indirect Biologic Indicators

The most useful diagnostic test for nitrate toxicity is a blood MetHb level.

This can accurately be determined using ABGs with co-oximetry (standard co-oximetry can differentiate MetHb from carboxyhemoglobin, oxyhemoglobin and deoxyhemoglobin; newer generation co-oximeters expand this detection capability).

Likewise, multiple wavelength pulse-oximeters exist that can noninvasively and continuously determine MetHb levels. Some newer generation multiple wavelength pulse oximeters can distinguish sulfhemoglobin from methemoglobin.

Key Points

  • Methemoglobinemia results in distinct changes in blood color and oxygen-carrying capacity.
  • Standard pulse-oximetry measurement of the oxygen saturation of hemoglobin does not provide accurate results in the setting of methemoglobinemia.
  • Oxygen saturation values from ABG analysis (without co-oximetry) is calculated based on a normal hemoglobin nomogram, rather than measured directly.
  • A "saturation gap" between the measured oxygen saturation of blood (standard pulse oximetry) and oxygen saturation calculated by routine blood gas analysis increases suspicion for methemoglobinemia.
  • Co-oximetry with ABGs is an accurate method of measuring MetHb levels and oxygen saturation.
  • Multiple wavelength pulse oximeters exist that can noninvasively measure and continuously monitor MetHb levels and oxygen saturation.

Progress Check

18. A drop of blood with MetHb appears as what color on filter paper?

A. Chocolate-brown.
B. Red.
C. Violet.
D. Clear yellow.


To review relevant content, see "Bedside Testing Instructions" in this section.

19. Of the following, which is the best method for measuring MetHb levels?

A. Standard ABGs.
B. ABGs with co-oximetry.
C. CBC with differential and blood smear.
D. Standard pulse oximetry.


To review relevant content, see "Standard Pulse-oximetry and Arterial Blood Gases" and "Co-Oximetry and MetHb Levels" in this section.

20. Clinically useful diagnostic test(s) for nitrate toxicity include

A. Measurements of nitrates in blood, urine, or saliva.
B. Measurements of nitrites in blood, urine, or saliva.
C. Blood MetHb level.
D. All of the above


To review relevant content, see Table 4.

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