Total body phosphate is found mostly in bone (80-85%), with smaller amounts in muscle and the extracellular fluid (<1%). Phosphate is the major intracellular anion. Terms associated with phosphate are confusing:

  • Phosphorus: The chemical element. Since it is unstable, phosphorous is never found free, but is always in the oxidized state (PO43-).
  • Phosphate: Phosphorus bound to oxygen (PO43-). This is found in the body as inorganic and organic phosphate
    • Inorganic phosphate: This is also called orthophosphate or the salt of the phosphoric acid. This form of phosphate is largely free “free” (not bound to any carbon-containing molecules) and is found in two forms with bound hydrogen:
      • This is the form that is measured in biochemical assays.
      • In the blood, it is largely “free” (not bound to anything) in two forms with bound hydrogen:
        • H2PO4: Diphosphate.
        • HPO42-: Monophosphate.
        • The ratio of these two forms varies depending on the pH. At physiologic pH, the ratio is 1:4 di:monophosphate, but when the pH falls (and H+ increases), the hydrogen binds to the monophosphate form, increasing the ratio. Recall that inorganic phosphate is one of the “unmeasured” anions that buffers increases in hydrogen (or acidosis).
      • Inorganic phosphate is also protein bound (around 10%) with some being complexed to positively charged molecules, including sodium, calcium and magnesium (5%). Some is also bound to lipoproteins.
    • Organic phosphate: Phosphate bound to a molecule containing carbon or a phosphate ester. Organic phosphate is in phospholipids, phosphate esters, phosphoproteins, nucleic acids, ATP, etc. This form is not measured in biochemical assays, but over time (e.g. hemolyzed samples that are stored, particularly at high temperatures such as 37°C), organic phosphate is cleaved by phosphatases to form inorganic phosphate (Tietz, Textbook of Clinical Chemistry, 3rd edition), which will be measured in our assay.


Serum or plasma inorganic phosphate only represents a small fraction of phosphate in the body, most of which is found in bone in the form of hydroxyapatite and calcium phosphate. Thus, serum or plasma inorganic phosphate is not a good reflection of total body stores, but it is considered a good marker of extracellular phosphate (i.e. that immediately available to cells), which only comprises about 1% of total body phosphate (the rest being in soft tissues, such as skeletal muscle). Phosphate is used in a lot of cellular processes. it is a key component of phospholipid membranes, metabolic proteins (e.g. ATP, glycolytic pathways) etc.

  • Release from bone: Phosphate is released by the action of parathyroid hormone (PTH), which concurrently releases calcium.  Bone release is promoted by acidosis, through the stimulation of PTH.
  • Absorption: Phosphate is absorbed in the small intestine (especially the jejunum, except in horses where the main sites of absorption are the colon, in particular, and  cecum) by both paracellular (passive diffusion) and active transport linked to sodium transporters (mostly Npt2b, with a lesser role of Pit1 and Pit2 [review by Sabbagh et al 2011]). Absorption is enhanced by low dietary calcium, increased dietary acidity, growth hormone and vitamin D. Absorption is decreased by low vitamin D, high calcium and low phosphate levels in the diet and other compounds such as iron and aluminum (e.g. antacids).
  • Excretion: Phosphate is primarily excreted in the urine (60-90%) in monogastric animals, with the rest being secreted into feces (30-40%). This differs in adult ruminants, in which phosphate is mostly excreted in saliva (where it used by rumen microbes and buffers rumen pH, but mostly is reabsorbed) and feces, with little excreted into urine (see excellent review on phosphate metabolism in ruminants by Grünberg 2014), except in young animals or adults on a high phosphate diet. Of course, milk is also a major route of excretion in lactating dairy cattle and the developing fetus (in the later stages of gestation) is also a source of phosphate “loss” to the dam. In the kidney, 80-90% of filtered phosphate is resorbed in the proximal convoluted tubule with sodium. The remainder is absorbed in the distal nephron. Renal excretion is increased by PTH, saline distension, phosphatonins, corticosteroids (which downregulate the sodium transporters on the luminal surface), catecholamines, calcitonin, bicarbonate and aciduria (in ruminants); whereas excretion is reduced by thyroid hormone, growth hormone, and insulin-like growth factor. Salivary excretion is also stimulated by PTH under conditions of normophosphatemia. The main mechanism for regulating phosphate in the body of monogastric animals is by regulating the amount excreted in the urine (this is primarily achieved via PTH and phosphatonins, such as FGF23).
  • Transcellular movement (translocation): This is not a mechanism of controlling body phosphate levels, but phosphate can move into or out of cells via sodium-dependent transporters in response to hormones or changes in pH.
    • Insulin: Cause phosphate movement into cells from plasma, decreasing serum/plasma phosphate. This also occurs with hyperglycemia, which stimulates insulin secretion. Insulin stimulates glycolysis, which requires ATP, so it makes sense that phosphate would move intracellularly.
    • Catecholamines: This is thought to stimulate uptake by cells.
    • pH: Alkalemia, e.g. due to respiratory alkalosis, will cause phosphate to also move into cells, due to the effect of alkalemia on stimulating glycolysis. Acidemia has the reverse effect on glycolysis, thus increasing extracellular or serum/plasma phosphate (Grünberg 2014).


The following colorimetric method with ammonium molybdate is used at Cornell University to measure inorganic phosphate.

Reaction type

Blanked end-point


In the presence of sulfuric acid, inorganic phosphate reacts with ammonium molybdate to form an ammonium phosphomolybdate complex, whose presence is detected photometrically in the ultraviolet region at 340 nm. The amount of ultraviolet light absorbed by the complex is directly proportional to the concentration of inorganic phosphate.

Reaction shown below:

Inorganic Phosphate + Ammonium molybdate sulfuric acidAmmonium phosphomolybdate

Units of measurement

The concentration of phosphate is measured in mg/dL (conventional units) and mmol/L (SI units). The conversion formula is shown below:

mg/dL x 0.3229 = mmol/L

Sample considerations

Sample type

Serum, plasma, and urine. Note that, although inorganic phosphate can be measured in other samples (saliva, RBC lysates, feces, muscle biopsies, bone biopsies, rumen), serum or plasma phosphate remains the gold standard (albeit not a precise one) for evaluating body (or more accurately extracellular) phosphate (Grünberg 2014).


Oxalate, EDTA and citrate anticoagulants can apparently result in spurious increases of phosphate concentration; therefore, heparin is the only suitable anticoagulant for testing phosphate levels. Internal studies in the Clinical Pathology Laboratory at Cornell University in bovine blood show that values in heparinized plasma are similar to serum (Naeves and Stokol, unpublished data), although previous studies have shown that values are lower in plasma (Grünberg 2014).


With prolonged storage, organic phosphate may leak out of cells (which develop permeable membranes due to reduced ATP) and increase in serum and plasma if not separated from cells. This may occur with (usually) or without overt hemolysis (red blood cells are found in the highest concentration in blood and intracellular organic but not necessarily inorganic phosphate are high), but requires organic phosphate to be converted to inorganic phosphate by phosphatases.

  • Human: Per reagent manufacturer product information sheet
    • Serum and plasma: 24 hours at room temperature, 4 days refrigerated, and 1 year frozen (-20°C).
    • Urine: 6 months refrigerated when acidified, and 3 weeks frozen (-20°C).
  • Bovine: Internal studies in the Clinical Pathology Laboratory at Cornell University show that there are minimal differences in phosphate results (i.e. changes of 0.2 mg/dL or less) in whole blood stored in a red top or green top (heparin) tube (before separation) for up to 6 hours at 4 or 22°C (Naeves and Stokol, unpublished data).


  • Lipemia, icterus: No effect
  • Hemolysis: Hemolysis apparently releases intracellular organic phosphate in red blood cells, which is cleaved with storage (particularly at 37ºC, but may also occur at room temperature) by phosphatases to inorganic phosphate. Thus, stored hemolyzed samples may have falsely increased phosphate concentraitons. Since inorganic phosphate is reportedly lower in human red blood cells than serum/plasma (Tietz, Textbook of Clinical Chemistry, 3rd ed.), in vivo intravascular hemolysis or in vitro hemolysis (preanalytical artifact) alone will not result in increased phosphate concentration (unless a blood sample from a patient with intravascular hemolysis or in vitro hemolysis is stored inappropriately, i.e. not separated from cells, not refrigerated and for some time before analysis). Indeed, we have not observed any clear correlation between the hemolytic index (degree of hemolysis, in vitro or in vivo) and phosphate concentrations in sick or healthy animals (internal studies done at Cornell University). In one study, there were minimal changes in phosphate concentration in freshly spiked pooled serum samples from dogs, cats, horses and cattle (Jacobs et al 1992).


Due to salivary excretion, values of phosphate in jugular venous blood can be lower (up to 19%) than in tail vein, udder or arterial samples in cattle (the decrease is attributed to drainage of phosphate from blood into salivary tissue) (Grünberg 2014).

Test interpretation

Increased phosphate concentration (hyperphosphatemia)

Increased serum phosphate indicates an increase in inorganic phosphate, which is in essence an acid (or the anion bound to hydrogen), because it is bound to hydrogen. Therefore an increase in serum phosphate indicates a hyperphosphatemic acidosis via strong ion principles. If the increase in phosphate is high, it can precipitate with calcium causing metastatic mineralization (mineralization of normal tissue) leading to deleterious effects on various organs in which the mineralization occurs (kidney, gastrointestinal system). This most commonly occurs with in small animals with chronic renal disease and vitamin D toxicosis (which is rare). The most common pathologic cause cause of high phosphate in small animals is decreased glomerular filtration rate (prerenal, renal or post-renal causes), whereas a high phosphate is uncommon in ruminants and frequently due to dehydration.

  • Artifact
    • Hemolysis: Hemolysis apparently results in release of organic phosphate from red blood cells into serum/plasma, which may falsely increase inorganic phosphate concentrations, but requires prolonged sample storage and temperatures higher than 4°C (see above).
    • Monoclonal gammopathy: A pseudohyperphosphatemia is a rare occurrence in animals with monoclonal gammopathies, which was attributed to the binding of phosphate to the monoclonal protein (Kristensen et al 1991). This artifact is likely analyzer dependent.
  • Physiologic: A mild post-prandial hyperphosphatemia may occur after the consumption of a meal, however a recent study showed that the increase was mild and concentrations remained within the reference interval for 46 of the 100 clinically healthy dogs with increased concentrations. The peak median increase was seen 8 hours after eating (Yi et al 2022). Young animals can have higher phosphate than older animals. This is at least in part due to the increased growth hormone, which increases renal tubular resorption of phosphate.
  • Iatrogenic: Administration of phosphate-containing fluids or compounds, e.g. sodium phosphate enemas, or high phosphate diets. Use of hypertonic sodium phosphate enemas in cats has been recognized to cause extreme hyperphosphatemia through absorption of phosphate. Hypernatremia, hypokalemia, hypocalcemia, and high anion gap metabolic acidosis are additional abnormalities. Affected cats can die. Morbidity and mortality resulting from use of these enemas is increased by prolonged retention of the hypertonic phosphate, mucosal defects enhancing absorption, and pre-existing abnormalities in water and electrolyte balance (Atkins et al 1985Jorgensen et al 1985).
  • Pathophysiologic: As indicated above, a decreased glomerular filtration rate is the most common cause of high phosphate.
    • Increased intake:
      • Hypervitaminosis D: This promotes absorption of phosphate and calcium in the intestine, resulting in hyperphosphatemia and hypercalcemia. This can be due to ingestion of vitamin D containing plants (e.g. Solanum, jessamine, golden oat in horses) or ingestion of vitamin D containing rodenticides. Because both calcium and phosphate are increased, metastatic mineralization and renal failure can be a consequence of these toxicoses.
      • Excess phosphate in diet: Diets that are high in phosphate (and low in calcium) can increase serum phosphate and result in secondary nutritional hyperparathyroidism (e.g. bran or “big head” disease in horses [Denny 1985]). High dietary phosphate can disrupt calcium homeostasis (particularly in transition cows) and can predispose to urinary calculi formation in cattle (Grünberg 2014).
    • Transcellular shifts
      • Acute tumor lysis syndrome: This results in high phosphate, high potassium, high uric acid and low calcium. Animals often die of acute oliguric renal failure.
      • Severe soft tissue trauma: This can also result in increased phosphate as phosphate is higher intracellularly in muscle than extracellularly, e.g. rhabdomyolysis. This would only really be expected with severe skeletal muscle injury (and not smooth or cardiac muscle) because skeletal muscle has such a large mass. In contrast, smooth muscle does not have as much mass as skeletal muscle and cardiac muscle injury severe enough to cause hyperphosphatemia will usually result in death.
    • Decreased excretion: The main route of excretion is the kidney.
      • Decreased GFR due to renal or post-renal azotemia: This is the most common cause of hyperphosphatemia in small animals. Many small animals that have a renal or post-renal azotemia are hyperphosphatemic, particularly with acute kidney injury and later stages of chronic renal disease (phosphate concentrations may be normal in early chronic kidney disease in small animals). The converse (hypophosphatemia) is often true for large animals, although some animals with renal or post-renal azotemia will also be hyperphosphatemic. Horses with renal azotemia due to chronic renal disease and ruminants with post-renal azotemia due to urolithiasis often have low versus high phosphate (mechanism unknown in horses, postulated salivary excretion of phosphate offsets the increase in ruminants). A pre-renal azotemia from severe hypovolemia may also increase phosphate concentrations in all species, but concentrations may be normal with mild azotemia from prerenal causes (mild dehydration or hypovolemia). Dehydrated animals with no increases in urea nitrogen or creatinine would not be expected to have high phosphate concentrations through decreased GFR.
      • Dehydration in ruminants: This can increase phosphate due to reduced salivary excretion, which can be worsened by reduced GFR in hypovolemic states (Grünberg 2014).
      • Hypoparathyroidism: Lack of PTH results in retention of phosphate but loss of calcium via the kidneys.
      • Acromegaly: Growth hormone promotes retention of phosphate in the kidney.
      • Hyperthyroidism: Phosphate is increased in up to 21% of hyperthyroid cats, presumably through this mechanism (thyroid hormone impedes urinary excretion).

Decreased phosphate concentration (hypophosphatemia)

Phosphate is an essential component of ATP, the energy source of the cell. Mild to moderate decreases in phosphate are common and are of minimal significance. Severe hypophosphatemia can be detrimental to the overall health of an animal. The following are clinical signs of severe hypophosphatemia:

  • Central nervous system: Seizures, coma and ataxia.
  • Muscles: Ileus in the gastrointestinal system and cardiomyopathy may result.
  • Kidney: Metabolic acidosis due to impaired bicarbonate absorption and calciuria result (with bone lysis).
  • Hematologic effects: Intravascular hemolysis is the most severe side-effect but is quite rare. ATP is required for normal red blood cell membrane integrity. An intravascular hemolytic anemia can occur when serum phosphate is < 1.0 mg/dL in dogs and < 1.5 mg/dL in cats. This is an important complication (life-threatening) of therapy for diabetic mellitus. Diabetes mellitus results in whole body phosphate depletion due to osmotic diuresis and decreased muscle mass. Therefore, diabetic animals often have total body phosphate depletion, regardless of serum or plasma phosphate levels (which are not good reflectors of total body phosphate). Insulin therapy causes phosphate to shift intracellularly, resulting in severe hypophosphatemias within 24-36 hours of administration. Phosphate levels should be monitored in diabetic animals treated with insulin. The syndrome of post-parturient hypophosphatemia with intravascular hemolytic anemia has been recognized in a few high-producing dairy cows fed low phosphate rations (Grünberg 2014). Hypophosphatemia also decreases neutrophil function and platelet survival.

Causes of low phosphate: Mechanisms can be multifactorial (and frequently is) in a given animal.

  • Artifact: Phosphate may be spuriously low if it precipitates out of solution. This can occur in the presence of monoclonal immunoglobulins with some phosphate reagents and analyzers (Kerr et al 2007).
  • Iatrogenic: Phosphate-binding antacids will decrease absorption.
    • Drugs: Drugs causing diuresis or promoting phosphate loss can result in hypophosphatemia, e.g. diuretics, corticosteroids. Sodium bicarbonate administration will promote renal losses of bicarbonate as well.
  • Pathophysiologic: Low phosphate is not a frequent finding in small animals. In large animals, a low phosphate can be seen in horses with chronic renal disease and gastrointestinal disease (decreased absorption, increased loss). Low phosphate can be seen in frequently in sick cattle, but the cause has not been clearly defined (it has been attributed to decreased feed intake [Grünberg 2014]).
    • Deficient intake/absorption: Gastrointestinal disorders affecting the small intestine may result in malabsorption of phosphate.  Low phosphate in cattle with displaced abomasa was attributed to anorexia in one study (Grünberg et al 2005), however there have been no controlled studies to measure phosphate concentration in which cattle were deprived of food to support this conclusion. In ruminants, low phosphate diets can result in low phosphate (Little 1984).
      • Enteral nutrition: Phosphate decreases 12-24 hours after enteral tube feeding in cats and may induce intravascular hemolysis.
      • Vitamin D deficiency: Vitamin D is required for phosphate absorption. This is a rare cause of hypophosphatemia.
    • Decreased release from bone: This can occur in cattle with post-parturient milk fever. The hypocalcemia in these animals can alter the setpoint for PTH responsiveness in bone, making the bone more resistant to PTH’s action (so there is less osteolysis and less calcium and phosphate is released in response to PTH). Phosphate losses through the milk and fetus contribute to the low phosphate, which can result in an intravascular hemolysis (called postparturient hemoglobinuria) if severely decreased (<1.0-1.5 mg/dL). However, this phenomenon is rare and has been called into question (Grünberg 2014).
    • Transcellular shifting
      • Alkalemia due to respiratory alkalosis: This causes a decrease in blood pCO2 which increases intracellular pH. The increase in pH stimulates phosphofructokinase activity which enhances glycolysis and phosphate incorporation into ATP, causing phosphate to move into cells to supply the enhanced phosphorylation that results (likely along the concentration gradient established when phosphate is used intracellularly for glycolysis). Respiratory alkalosis occurs with hyperventilation such as secondary to sepsis, heat stroke, central nervous system problems, hepatic coma, salicylate toxicity and fear. Similarly, the post-prandial alkaline tide that occurs after eating may result in mildly decreased phosphate post-prandially.
      • Catecholamines: These can stimulate glycolysis and thus induce intracellular movement of phosphate (Liamis et al 2010). This is one of the postulated mechanisms for hypophosphatemia in septic patients (Chu et al 2021).
      • Insulin or glucose administration. Hyperglycemia stimulates insulin secretion, which causes glucose to move into cells and be used for ATP production. Since this consumes phosphate in the cells and lowers intracellular phosphate concentrations, phosphate moves into cells along the created concentration gradient (lower in cells, higher in plasma). In states of insulin resistance, this would not be expected to occur.
    • Increased loss: This usually occurs through the kidneys or gastrointestinal system. Note that high producing dairy cattle also lose phosphate in the milk and for the developing fetus.
      • Renal losses:
        • Hyperparathyroidism: Phosphate is expected to be low in primary hyperparathyroidism due to increased renal excretion of phosphate secondary to high PTH. However, phosphate levels may be normal or elevated if there is a concurrent decrease in glomerular filtration rate (GFR) and we have seen normal phosphate concentrations in animals without alterations in GFR. The combination of high calcium and low phosphate is typical for hyperparathyroidism in small animals. Increases in PTHrP as a consequence of malignancy may also result in hypophosphatemia (PTHrP works via PTH receptors) but this is usually not seen. Rather affected animals usually only have hypercalcemia.
        • Renal disease: Since phosphate is resorbed in the renal tubules, proximal renal tubular acidosis can result in loss of phosphate. Hypophosphatemia is a characteristic (but not invariably present) finding in horses with chronic renal disease. In horses, the combination of high calcium (from decreased renal excretion) and low phosphate is typical of chronic renal disease but can also be seen in acute renal injury. The mechanism of hypophosphatemia is unclear in horses with renal disease (perhaps increased phosphatonins, like FGF23?).
        • Urolithiasis in ruminants: In cattle and goats with post-renal azotemia due to urolithiasis, a low phosphate is typical versus high phosphate (normally, we would expect to see a high phosphate with decreased GFR from post-renal azotemia). This is attributed to increased secretion of phosphate in saliva (which is then degraded in the gastrointestinal tract), which prevents an increase in serum phosphate in these species (George et al 2007).
        • Osmotic or solute diuresis: Polyuria from osmotic diuresis will result in renal phosphate losses, e.g. diabetes mellitus. The lack of insulin (which would normally cause movement of phosphate into cells) may normalize serum phosphate levels, despite total body depletion of phosphate.
        • Inhibitors of renal resorption: Tumoral osteomalacia results when some tumors, especially mesenchymal tumors, release inhibitors of phosphate reabsorption. These inhibitors are called phosphatonins, one of which is FGF23 (fibroblast growth factor 23). This protein inhibits absorption of phosphate in the kidney, promoting phosphaturia, and decreases vitamin D (hence the osteomalacia mimics that seen with vitamin D deficiency, or rickets). High concentrations of FGF23 are seen in cats with chronic kidney disease and some soft tissue sarcomas in dogs (Hardcastle and Dittmer 2015).
        • Acidemia/acidifying diets: Renal tubular absorption of phosphate is decreased with aciduria (expected as a compensatory or corrective response to acidemia) in ruminants, which may result in low phosphate (Grünberg 2014).
        • Cushing’s disease in dogs: Hyperadrenocorticism can result in hypophosphatemia because corticosteroids promote renal loss of phosphate. It is unknown if this occurs in horses with pars pituitary intermedia dysfunction (PPID) (many of these horses have high levels of ACTH and cortisol as well).
      • Gastrointestinal losses: Vomiting, diarrhea.
      • Other sources of loss: Both the developing fetus (in late pregnancy) and colostrum and milk (particularly with high demands just after calving) require phosphate, resulting in decreased phosphate in blood in the postpartum period. Concurrent hypocalcemia can worsen the hypophosphatemia because it will stimulate PTH release, which promotes additional phosphate loss through the kidney and gastrointestinal system (saliva). Correction of hypocalcemia frequently corrects the low phosphate (by decreasing PTH and restoring gastrointestinal motility, which allows dietary and salivary phosphate to be reabsorbed in the gut). However, some cows do not recover with correction of hypocalcemia and can remain as “downer” cows and have very low phosphate (<1 mg/dL). The mechanisms for this continued low phosphate is unclear, however such cows are at risk of postparturient hemoglobinuria (intravascular hemolysis with hemoglobinemia and hemoglobinuria because of phosphate depletion – remember ATP which contains phosphate is required for maintaining integrity of the RBC membrane, so RBCs can lyse with severe phosphate deficiency). This hemolytic disorder can occur in the first 6 weeks after calving. Current thoughts are that low phosphate alone does not result in this hemolytic syndrome and that affected cows are also depleted in other minerals (copper, selenium) and energy. Luckily, postparturient hemoglobinuria is rarely seen anymore.
    • Unknown mechanisms:
      • Hepatic lipidosis in cats.
      • Hypothermia: This can result in low phosphate and potassium concentrations, high calcium and glucose concentrations, metabolic acidosis and azotemia. One study in humans with severe head trauma suggested that hypothermia causes urinary phosphate losses through induced diuresis and this occurred as the core temperature was cooled to 32ºC (Polderman et al 2001). However, the authors did not measure fractional excretion of phosphate. Another study found no changes in urine output in brain-injured patients and did associate hypophosphatemia with hypokalemia and a higher heart rate, again during the cooling process. The authors speculated the hypophosphatemia was due to transcellular (intracellular) shifting of phosphate and potassium secondary to catecholamine release (Aibiki et al 2001).
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