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Serum/plasma Chemistry

Serum quality

Serum quality

Serum chemistry data may help determining the function of certain organ systems. The reliability of the results largely depend on the quality of the sample. Before running any biochemistry tests, the blood should be well clotted, preferably without hemolysis (shown as red coloration of the serum). Chemistry data are usually obtained from serum. Some tests can also be run using plasma. Before using plasma, this option should be checked with the test instructions.

 

Test tubes to be used: red-topped serum-tubes with or without a clotting activator.

 

The color of the serum should be light yellow. The figure below shows different serum characteristic:

Hemolysis:

  1. Red to brown color due to hemolysis (destruction of erythrocytes). This can have a pathological origin or can result from poor sampling/handling; the red-brown color can also be caused by myoglobin after massive muscle damage (rhabdomyolysis)

  2. Milky white color due to presence of fat particles in the serum (physiologic shortly after eating or pathological condition)

  3. Yellow color due to the presence of bilirubin (liver damage ->icterus). 

  4. Blueish-red color due to methemoglobin (low venous oxygen saturation).

Schematic drawing of tubes with blood. Note that the serum has different colors, depending on the abnormality in the animal: hemolyzed, lipemic, icteric and methemoglobin containing serum.

Lipemia

Lipemia, which can be a natural occurrence if the elephant has just eaten, can alter several test results. Calcium, phosphorus, total bilirubin or hemoglobin may be falsely elevated. When using a refractometer to measure the total protein remember that the serum must be clear. If not the value may be falsely elevated. Albumin, sodium and potassium may be falsely lower. Lipemia also enhances hemolysis which in turn can affect lab results. But there is a solution:  If you refrigerate your sample the lipemic portion will separate and you can use the clear aliquot below the lipid layer.

Test tubes containg non-lipemic and lipemic serum

Normal and lipemic elephant serum

Reference values

Normal serum chemistry values are determined by species, age, gender and reproductive status of the elephant. It is important to have an understanding of the limitations of laboratory values. The term “reference value” is now considered a more appropriate term than “normal value.” Ideally reference values should be established from studies using a minimum of 30 healthy animals and stated selection criteria. Few elephant studies have been conducted to meet this standard.

One report describes chemistry results for different genders of Asian elephants used for logging in Myanmar (Santo, 2020).

Moreover, many reference ranges are laboratory specific. So it is best to use one laboratory that can help to develop reference ranges for your elephants.

 

Another important point is that a test result that falls outside of the reference is not necessarily clinically significant. Lab values are information that must be used with all the other information that you have when you are faced with a sick elephant. Establishing a baseline during health and performing sequential tests during illness will give the most reliable information. You also want to use a lab that has good quality controls. If you change labs it is advisable to get new healthy baselines. Very odd results should always be double checked at the same lab.

Test result units

Another complicating factor when looking at serum chemistry values is that there is a lack of uniformity regarding units and this can be confusing. Most U.S labs use conventional units whereas in Europe they use SI units. There are conversion factors to go from one system to the other but the conversion factor is test specific – so each test has a different conversion factor. You can find SI conversion calculators online, i.g. https://www.amamanualofstyle.com/page/si-conversion-calculator.

Liver

The liver plays an important role in the following processes:

  • Protein synthesis and degradation (albumin, clotting factors)

  • Carbohydrate and lipid metabolism

  • Breakdown of hemoglobin

  • Storage (fat soluble vitamins)

  • Detoxification

 

Liver enzymes:

  • Aspartate aminotransferase (AST; SGOT)

  • Alkaline phosphatase (ALP)

  • γ-glutamyl transferase (GGT)

  • Bilirubin

  • Bile acids (?)

  • BSP excretion (bromsulphthalein)

 

AST (SGOT)

Aspartate aminotransferase, previously known as serum glutamic oxaloacetic transaminase (SGOT) occurs in all cells. Highest levels are in the liver, cardiac muscle, and skeletal muscle. If AST is elevated then you should also look at the creatine kinase (CK) value.  If the CK is normal then AST is likely of liver origin. If the CK is elevated or there is obvious muscle trauma then AST may be of muscle origin. Also AST may falsely increase if the sample is hemolyzed.

 

ALP

Alkaline phosphatase is also found in all cells with the highest levels in liver, bone, kidney, intestine, and placenta. ALP is not a sensitive indicator of liver disease in the horse and this probably holds true for elephants although research would be needed to confirm this assumption.  ALP may increase with disorders such as rickets. ALP levels are normally higher in young animals including elephants. ALP may indicate colostrum absorption. Non-steroidal anti-inflammatory drugs may cause ALP to elevate.

 

GGT

Gamma glutamyl transferase (GGT) is liver specific in horses and pigs. Whether it is liver specific in elephants is unknown. GGT is an indicator of cholestasis (the interruption of bile excretion). GGT has been shown to increase in musth bulls and it has been used together with ALP as an indicator to evaluate passive transfer of antibodies to neonates via colostrum.

 

Bilirubin

There is not much information about bilirubin in elephants. There are two forms: unconjugated and conjugated bilirubin. The unconjugated is the main form in horses so this may be true for elephants but we don’t know for sure.

Unconjugated (indirect) bilirubin is mainly hemoglobin released from old erythrocytes. It is bound to albumin and transported to the liver where it is conjugated. Conjugated (direct) bilirubin is secreted into bile, transferred to the intestine, converted to urobilinogen by intestinal bacteria, and excreted. Elevated bilirubin may be caused by hemolysis; hepatocellular disease that results in reduced functional mass; and intra - or extrahepatic cholestasis or bile duct obstruction. Unconjugated bilirubin predominates in horses with hyperbilirubinemia regardless of etiology whereas in ruminants unconjugated bilirubin is typical.

In one report, elevated total bilirubin (4.94 mg/dl) was observed in a female Asian elephant with colic caused by over-zealous feeding of produce. Values for indirect and direct bilirubin were 3.7 mg/dl and 1.2 mg/dl respectively. 

Schematic drawing of the hepatic uptake, conjugation, and secretion of bilirubin. Diagram from Lattimer, K.S., Mahaffey, E.A., and Prasse, K.W. 2003.  Clinical Pathology 4th edition. Blackwell. P.199.

Tests of hepatic uptake, conjugation and excretion of bilirubin. Diagram from Lattimer, K.S., Mahaffey, E.A., and Prasse, K.W. 2003.  Clinical Pathology 4th edition. Blackwell. P.199.

Bile acids

Bile acids assist with fat digestion. In most species, bile acids are stored in the gall bladder and released into the intestine. However, elephants do not have a gall bladder. There is controversy whether elephants have bile acids. In several cases bile acids were shown to increase in elephants with TB.

 

Bromsulphthalein (BSP) excretion test

In the BSP test a dye is injected IV and measured at several points in time post-injection. In the horse, the half-life is 3.5 minutes. Slow clearance time may indicate cholestasis. Although levels have been shown to increase with liver flukes in elephants, it is not a very practical test.

Kidney

 

Blood Urea Nitrogen (BUN) and creatinine are the main enzymes used to evaluate kidney function in mammals.

 

BUN

Elevations in blood urea nitrogen concentration may be due to prerenal causes like inadequate renal perfusion, shock, or diminished blood volume; renal causes like glomerular-nephritis; or postrenal causes like urinary tract obstruction.

Blood Urea Nitrogen makes up approximately 75% of the total non-protein nitrogen (NPN) fraction of the blood. BUN is the major end product of protein nitrogen metabolism. It is synthesized by the urea cycle in the liver from ammonia which is produced by amino acid deamination. Urea is excreted mostly by the kidneys, but minimal amounts are also excreted in sweat and degraded in the intestines by bacterial action.

 

Creatinine

Creatinine only elevates when disease is severe and there is marked kidney damage. Unfortunately, in elephants these enzymes are not always useful to predict kidney disease.

Creatinine may be lower in young elephants; higher in musth bulls.

 

Serum osmolality/urine osmolality.

The osmolality reflects the total number of electrolytes in a fluid. To keep the number constant, the kidney excretes the surplus that is present in the liquid fraction of the blood and the osmolality ratio between these two fluids should be <1. If the kidneys fail to maintain this equilibrium, the serum-urine osmolality ratio becomes > 1.  

Serum dimethyl arginine (sDMA).

In domestic animals sDMA is a marker for endothelial dysfunction and early diagnose of renal disease (declining glomerular filtration rate). In one case report it was associated with kidney failure (polycystic kidney disease).

Blood chemistry values from an Asian bull elephant with capture myopathy. The elevations in SGOT and CK are dramatic. ALT (SGPT) did not increase as much but is probably significant. The elevations in BUN and creatinine may have been related to capture myopathy however this bull was chained to a tree and not given access to food or water post-capture so these changes may reflect dehydration.

These values are from an Asian bull elephant with capture myopathy. The elevations in SGOT and CK are dramatic. ALT (SGPT) did not increase as much but is probably significant. The elevations in BUN and creatinine may have been related to capture myopathy however this bull was chained to a tree and not given access to food or water post-capture so these changes may reflect dehydration.

Muscles

Muscle enzymes are:

  • Creatine Kinase (CK)

  • Lactate Dehydrogenase (LDH)

  • Aspartate Aminotransferase (AST; SGOT)

  • Alanine Aminotransferase (ALT; SGPT)

 

Evaluating muscle enzymes can help to diagnose muscle pathology. Conditions that may cause elevated muscle enzyme levels include prolonged recumbency, rhabdomyolysis  (also called over-exertion, or tying up syndrome), and clostridial myositis. Bacterial endocarditis, and aortic thrombosis are other causes as well as Vit E/Se deficiency and capture myopathy. Working elephants may be at risk for muscle over-exertion disorders especially as the planet heats up. In all of these conditions, muscle cell membranes rupture and enzymes are released into the blood.

Conditions that may lead to increased muscle enzymes in serum are:

  • Prolonged recumbency

  • Rhabdomyolysis

  • Clostridial myositis

  • Bacterial endocarditis

  • Aortic thrombosis

  • Vitamin E/ selenium deficiency

  • Capture myopathy

 

Creatine kinase (CK)

CK is critical to muscle energy production. Highest levels are in skeletal muscle, cardiac muscle, and brain. Most CK in the serum is of muscle origin and it is the most sensitive indicator of muscle damage. CK rises quickly – within hours. It also returns to normal quickly as long as there is no on-going damage. Levels that remain high indicate an on-going disease process. Hemolysis interferes with the test and causes falsely elevated values.

 

Lactate dehydrogenase (LDH)

LDH is present in all tissues. Muscle, liver, and red blood cells are the usual sources. LDH is not as useful for determining muscle damage because it is not muscle specific. If LDH is elevated and there is no muscle injury then liver problems should be considered. Like CK, LDH will be falsely elevated in the presence of hemolysis.

 

Aspartate aminotransferase (AST)

AST was previously known as serum glutamic oxaloacetic transaminase (SGOT).  Muscle and liver are the major sources. It is another enzyme to check if muscle damage is suspected. 

 

Alanine aminotransferase (ALT)

ALT, also known as serum glutamic pyruvic transaminase or SGPT, is considered muscle specific in large domestic animals. Increased levels have been associated with myopathies in a number of species. ALT will increase in recumbent elephants that are down for a long time.

 

Severe muscle damage can occur in case of Capture Myopathy:

Calcium

Ionized calcium

This electrolyte that is involved in many chemical reactions in the body. The active form is ionized calcium (Ca2+) and this parameter gives the best impression of the available calcium. To measure ionized calcium, special heparinized tubes are needed. Ionized calcium should be > 1.5 mmol/L (>5 mg/dL). Low ionized calcium levels are associated with prolonged parturition and dystocia.

 

Total calcium

Non-ionized calcium is predominantly conjugated with albumins and expressed as total calcium.

Higher levels of total calcium have been reported in very young elephants and in elephants with TB and severe kidney disease.

Many pregnant captive elephants develop subclinical hypocalcemia if calcium is not supplemented during pregnancy.  When parturition starts, the demand of calcium is high as it essential for uterine contractions that open the cervix and help expel the calf. Calcium is also high in milk. Calcium supplementation during pregnancy is recommended to form a stock supply. However, releasing stock-calcium ions from the bones is a very slow process, so the blood itself should contain enough calcium conjugated to albumin to supply the uterus and body muscles for these hours of high demand.

Total calcium levels lower than 1.5 mmol/l (6 mg/dl) result in recumbency and levels less than 1 mmol/l (4 mg/dl) will result in death. Low total calcium levels are associated with prolonged parturition and dystocia. Total calcium should be > 2.8 mmol/L (>11 mg/dL).

Other minerals and electrolytes

Sodium (Na)

Potassium (K)

Chloride (Cl)

The reference ranges of these electrolyte in elephants are similar to other species and can best be compared with horse values.

Proteins

Elephants have a higher total protein serum level than most mammals; albumin is lower, and globulins are higher. The ratio of albumin to globulin was shown to be lower in one study that compared elephants with and without TB however the number of TB positive elephants in the study was low.

Protein electrophoresis separates the albumin and globulin fractions. There are not many reports in elephants. Protein electrophoresis may be useful to monitor inflammatory conditions or problems like TB, herpes, and others.

Acute Phase Proteins

Acute phase proteins (APPs) are components of the innate immune system that are markers for infection, inflammation, neoplasia, and tissue injury in humans and domestic animals. APPs are produced by the liver in response to cytokines released from leukocytes. They are initially released into serum 24–96 hr following an acute inflammatory stimulus, where they function to promote healing, reestablish homeostasis, and inhibit microbial growth. The main ones are C-reactive protein (CRP), serum amyloid (SAA) and haptoglobin (HA). In one study serum samples from 35 healthy Asian elephants were analyzed for these 3 APPS and levels between the values in healthy and unhealthy elephants were compared (Isaza et al., 2014). From this and other studies it seems that SAA may be the most responsive APP in elephants. The APP values in this study in Asian elephants are shown here:

C-reactive protein          12.4-122 nmol/l (1.3-12.8 mg/l)

Serum amyloid               0-47 mg/l

Haptoglobin                   0-1.1 mg/l

Glucose

Glycolysis of glucose in serum or plasma will decrease the glucose level starting shortly after blood collection. Serum or plasma should therefore be separated from the red blood cells within 30 minutes after collection. Glycolysis can be prevented by using a Na-fluoride tube for blood collection. Significantly lower levels have been noted in one study comparing TB culture positive and negative elephants. In horses, hypoglycemia may be seen with hepatic failure or bacteremia. There is one report of diabetes mellitus in a 50-yr-old Asian elephant (van der Kolk 2011).

Hyperadrenocorticism and hyperthyroidism have not been reported in elephants. Increased blood glucose in elephants is likely to be transient. Some of the causes of transient hyperglycemia could be acute severe colic

acute stress, post-postprandial, certain drugs (steroids, xylazine, phenothiazine).

Hypoglycemia can occur in neonatal elephants and in cases associated with malnutrition, malabsorption, sepsis, extreme physical exertion, advanced liver disease or neoplasia.

Amylase and lipase

Ranges for these enzymes vary tremendously depending on the methodology and the lab so at this point in time they are not very useful tests for elephants.

Amylase may increase in case of pancreas, gastro-intestinal, liver and kidney disease

Lipase may increase in case of pancreas or kidney disease. One case of pancreatitis in an elephant has been described so far (pers. comm. Susan Mikota, 2023).

A comprehensive elephant serum chemistry panel should include:

  • Total protein

  • Albumin

  • BUN

  • Creatinine

  • AST (sGOT)

  • ALT (sGPT)

  • GGT

  • Bilirubin

  • Bile acids (?)

  • CK

  • LDH

  • Na

  • Cl

  • K

  • Ca

  • P

References

  • Steyrer C, Miller M, Hewlett J, Buss P and Hooijberg EH (2021) Reference Intervals for Hematology and Clinical Chemistry for the African Elephant (Loxodonta africana). Front. Vet. Sci. 8:599387 

  • Santos DJ, Franco dos J, John, Nyein UK, and Lummaa VM. 2020. Sex differences in the reference intervals of health parameters in semi-captive Asian elephants (Elephas maximus) from Myanmar. J.Zoo&Wildl Med 51(1): 25–38

  • Debbie JG and Clausen B. 1975. Some hematological values of free-ranging African elephants. Journal of Wildlife Diseases, 11(1):79-82.

  • Isaza R, Wiedner E, Hiser S, Cray C. 2014. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus). J. of Vet. Diagn. Invest. 1-6

  • Perrin, KL, Kristensen AT, Gray C, Nielsen SS, Bertelsen MF, Kjelgaard-Hansen M. 2020. Biological variation of hematology and biochemistry parameters for the Asian elephant (Elephas maximus), and applicability of population derived reference intervals. Journal of Zoo and Wildlife Medicine, 51(3) : 643-651.

  • Van der Kolk JH,  Hoyer MJ, Verstappen FALM, Wolters SABI, Treskes M, Grinwis GCM and Kik MJL (2011). Diabetes mellitus in a 50-year-old captive Asian elephant (Elaphas maximus) bull, Veterinary Quarterly, 31:2, 99-101. Https://doi.org/10.1080/01652176.2011.585793

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