Urea Breath Test

What is a Urea Breath Test?
Urea breath test is a simple test conducted on a patient's breath to detect H. Pyloriinfection. It is widely recognized as the "gold standard" for in vivo diagnosis of H. pyloriinfection. Only 2 breath samples are needed, one breath sample at the beginning of the test before ingestion of the urea tablet and the other breath sample at 20 minutes after ingestion of urea tablet.



Feature & Benefits of the Urea Breath Test

	Otsuka's UBT	Serology (ELISA)	Stool (HpSA)	Endoscopy
Accuracy	98% sensitivity 98% specificity	85% sensitivity 79% specificity	93% sensitivity 93% specificity	90-95% sensitivity 100% specificity
Administration	Non invasive	Requires drawing blood	Inconvenient, requires handling of faecal sample	Requires invasive procedure
Consistent Results	Tests for active infection reducing the chance of false positives	Serological test (for H. pylori) is not accurate enough for use in routine clinical	Considerable lot-to-lot variation in tests	Careful sample preparation is necessary for optimal results
Scope of Test	Tests the entire gastric mucosa for active H. pylori infection	Inactive antibody testing is 17 times more likely to cause unnecessary treatment	Second patient consultation is required	Endoscopic biopsies only targets small areas of the stomach
Eradication Monitoring	Indicated for the post-treatment monitoring of H. pylori infection to confirm eradication 4 weeks following completion of therapy	Cannot confirm eradication	Must wait 6-8 weeks after therapy to confirm eradication	Requires second invasive procedure to confirm eradication

How to Carry Out ¹³C-Urea Breath Test?
Easy & Safe for Your Patients

• 4 simple steps
• Non-radioactive
• If samples are analyzed in clinics, results can be obtained within 25 minutes

Please always instruct the patient to exhale into the mouthpiece of the bag until it is BLOATED. Sufficient amount of air sample is crucial to obtain valid results. Insufficient air in the bag may produce invalid results which require a repeat collection. This will lead to reagent wastage and inconvenience to your patient.



*Patient in a fasting condition to promptly (within 5 sec) swallow one UBIT Tablet with 100mL of water without being crushed or chewed.

Easy & Safe for Your Staff

• No special training required
• Breath collection takes only 20 minutes
• Call for laboratory service provider to collect breath sample

How Safe is the Test? 
¹³C-Urea Breath Test is widely recognized as the easiest, safest and most reliable non-invasive diagnostic test by many gastroenterologists. Unlike ¹⁴C, ¹³C is a stable, non-radioactive isotope. About 1.1% of our body contains naturally occurring ¹³C. A normal daily food intake of an adult will contain about 3g of ¹³C. During the test, patient takes 100mg ¹³C-Urea, which only represents a further intake of ¹³C. The amount of urea (100 mg) taken during a breath test is only a small percentage of the average total body urea pool of 10g.

What is Needed to do the Test? 
Only breath bags and urea tablets are required. There is no additional mouthpiece necessary. Otsuka's breath bags are neatly designed and are disposable, presenting a cost effective and safe solution! There are no recyclable parts, hence , no worries about risk of infections or contaminations. Extremely stable breath bags, allow breath sample to be stable up to 10 weeks after sampling date. In addition, the "one-way air valve design" of the breath bags prevents sampling leakages and errors.

What should My Patient Do before the Test?

• Fast for at least 6 hours prior to test
• No smoking for 2 hours prior to testing
• Ensure compliance with drug therapy – see below:
  Antibiotics / Antibacterial
  These drugs must be stopped at least 4 weeks before Urea Breath testing:
  Amoxicillin (Amoxil, Moxam),Bismuth tricitrate (Denol), Clarithromycin (Klacid), Fasigyn (Trinidazole), Metronidazole (Flagyl), Tetracycline (Tetrex, Mysteclin, Achromycin), and any other antiobiotics.
  
  Proton Pump Inhibitors
  These drugs must be stopped at least 1 week before Urea Breath testing:
  Losec (Omeprazole), Somac (Pantoprazole Sodium Sesquihydrate), Zoton (Lansoprazole) and Nexiam.
  
  H2 Receptor Antagonists
  These drugs must be stopped at least 24 hours before Urea Breath testing.
  Cimetidine (Tagamet, Sigmetadine, Magicul), Famotidine (Amfarnax, Pepcid, Pepcidine), Nizatidine (Tazac) and Quick EzeRanitidine (Zantac, Rani 2).

Thalassaemia

By Prof. Datin Dr. G. Duraisamy    Clinical Consultant (Haematology)

Thalassaemia can be found in any population of people but is most common in the Chinese and Malays in Malaysia, people from the South East Asia, Mediterranean & the Middle East. In Malaysia it is seen in 3 to 5% of Malays, Orang Asli and Chinese. HbE is seen in Malays & Orang Asli 3 to 5%.

Normal adult haemoglobin (HbA) has 2 alpha globin chains and 2 beta globin chains. There are 2 types of Thalassaemia

• Alpha (α) thalassaemia - lack of alpha globin chains
• Beta (β) thalassaemia - lack of beta globin chains

Thalassaemics pass down the gene to their offspring & may present as

• thalassaemia trait or  - usually healthy and most go through life unaware of their status thalassaemia minor
• thalassaemia - occurs if two thalassaemia minors marry intermedia
• thalassaemia major   - occurs if two thalassaemia minors marry

Clinical Classification of α and β Thalassaemia

Form	Alpha α	Beta β
Minor (Hb normal)	Trait	Trait
Intermediate (Hb 6 to 9g/dl)	HbH HbH with HbCS	β thal intermedia HbE/β thalassaemia
Major (Hb < 5g/dl)	Hb Barts hydrops foetalis	Homozygous beta (β) thalassaemia major

There are 4 Alpha genes, 2 on each chromsome.

Types of alpha thalassaemia:

• α+ thalassaemia or silent alpha thalassaemia (minor / trait) - 1 gene missing 
• α° thalassaemia or alpha thalassaemia minor (minor / trait) - 2 genes missing on the same chromosome
• HbH disease - 3 genes deletion
• Alpha thalassaemia major (causes hydrops foetalis in Chinese)  - 4 genes missing

α° thal trait
When MCV is < 76fl, MCH < 26pg, TRBC > 5m. and Hb analysis is normal, α° thalassaemia trait is tested for by doing the following test:

• Parents FBC, MCV - one parent has MCV < 76fl, MCH < 26pg and Hb analysis is normal
• *S.Ferritin* - normal to high levels of storage iron
• *DNA studies for α° thalassaemia

Types of beta thalassaemia:

• Beta (β) thalassaemia trait / minor
• Beta (β) thalassaemia intermedia
• Beta (β) thalassaemia major

Malays: β+ (reduced βchains): 50% have mutation IVS 1-5 (G to C)
Chinese: β° (no β chains): 49.8% mutation 41-42 (-TCTT)

β thalassaemia minor / trait is not serious. People with thalassaemia minor are healthy and do not have any effects on health or length of life.

β thalassaemia major is serious and may occur when two thalassaemia minors marry and have children. Beta thalassaemia major suffers from severe anaemia and needs treatment (red cell transfusions monthly and chelating agent like deferral) all their lives. There is no cure for thalassaemia major. Bone marrow transplant for beta thalassaemia major, if successful, may result in normal Hb formation and there will be no need for transfusions. Gene therapy may one day be available to "cure" thalassaemia but it is not yet available.


Haemoglobinopathy
Is a point mutation with 1 amino acid substitution that changes the structure and function of the Hb molecule. Example of haemoglobinopathies are HbE, HbS, Hb Constant Spring (HbCS).

HbE has a point mutation at codon 26 of β globin chain with GAG changed to AAG changing amino acid from Glutamatic Acid to Lysine amino acid.
It is seen mainly in Malays (3.5-4.5%), Orang Asli & those in the Golden Triangle area.

The heterozygous HbE combined with β thalassaemia trait give rise to HbE/β Thal (Thal Intermedia)

HbS is a mutation at codon 6 of β globin chain: Here amino acid Glu is changed to Val.
HbS is rare in Malaysia.

Hb Constant Spring (HbCS) has a point mutation at stop codon of the α globin chain, changing amino acid TAA to CAA:

• This results in an extra long globin chain with an additional 41 amino acids making the normally 131 α globin chain to become 172 amino acids long.
• HbCS is seen in Malays and Chinese - not in Indians
• Homozygous, combined with α¹ or α^° thal it is HbH disease, thalassaemia intermedia.

Laboratory Tests for Diagnosing Thalassaemia

• FBP-Hb, PCV, MCV, MCH
  
  

  MCV < 76 fl MCH < 27 PBF: Microcytic,    Hypochromic

  ⇒

  Commonly due to    Thalassaemia or Iron deficiency

• Reticulocyte smear - for "H" inclusions for α° Thal
  
       
• Hb electrophoresis or HPLC - for β thal, HbE, HbS,HbCoSp, HbH,Hb Barts, HbNY, HbD
  
  
• S. Ferritin: High in Thalassaemia & multiply transfused persons
• DNA studies for α° thalassaemia

Additional Tests that should be done for Multiply Transfused are:

• RBC genotype at diagnosis,
• Screen for HBsAg, anti-HCV, anti-HIV initially & annually,
• LFT - for transaminases done initially & annually.

Thalassaemia is a public health problem in Malaysia.


Suggested Flow for Screening those with a Low MCV, MCH
HbCS - normal MCV, MCH
HbE - MCV < 80fl, MCH < 26pg



Cascade studies:
When a person is identified with thalassaemia, screening their blood relatives would identify more of those with thalassaemia.

Cascade Screening:
More cost effective than population screening. Screen the patient's parents, siblings, children and other blood relatives of those with thalassaemia. Spouse can be screened for thalassaemia to assess the risk for thalassaemia major.


Risk for Thalassaemia Major if

• α° thalassaemia marries α° thalassaemia or
• β thalassaemia trait marries a β thalassaemia trait and has children

Risk for Thalassaemia Intermedia if

• β thalassaemia marries HbE and has children
• HbCS marries α° thalassaemia trait and has children

Prevention of Thalassaemia
FBC - Hb near normal; MCV, MCH if low, MCHC high, high rbc

• S. Ferritin
• Hb electrophoresis / HPLC: HbA₂, HbF, HbE, HbS, HbCS, HbH
• Reticulocyte smear: "H" inclusions for α° thalassaemia
• DNA studies for α° thalassaemia
• Prenatal Diagnosis: foetal blood sampling, foetal DNA by chorionic villi sampling
• Counselling - marriage of traits!
• Give a verbal and written (laminated) result
  
  

Diabetes Mellitus - Targets for Control

By Prof. Dr. Leslie Charles Lai Chin Loy   Clinical Consultant (Clinical Biochemistry)  Diabetes mellitus is a pandemic and its prevalence is increasing worldwide with dire health consequences for those afflicted with the condition. Around 50% of diabetics die of myocardial infarction and 25% die of cerebrovascular accident. In addition, diabetes is the commonest cause of blindness, end-stage renal disease and amputations in the world. The risk of macrovascular (coronary heart disease, stroke and peripheral vascular disease) and microvascular (retinopathy, nephropathy, peripheral and autonomic neuropathy) complications can be reduced by treating blood glucose, lipids and blood pressure to target. At the 6th International Diabetes Federation Western Pacific Region Congress in Bangkok, 22nd till 26th October 2005, the 4th Edition of 'Type 2 Diabetes Practical Targets and Treatments' was launched. The targets for control are shown in Tables 1 and 2. Table 1. Targets for Control Parameter Target HbA1c <6.5% (DCCT-aligned assay) Blood Pressure <130/<80 mmHg Total Cholesterol <4.5 mmol/L (<174mg/dl) LDL Cholesterol <2.5 mmol/L (<97 mg/dl) HDL Cholesterol >1.0 mmol/L (>39 mg/dl) Triglycerides <1.5 mmol/L (<133 mg/dl) Urinary albumin:creatinine <2.5 mg/mmol in men <3.5 mg/mmol in women Table 2. Recommended Targets for Glycaemic Control HbA1c Fasting plasma glucose / prepandial plasma glucose 2 hour postprandial plasma glucose <6/5% (DCCT-aligned assay) 4.4-6.1 mmol/L (80-110 mg/dl) 4.4-8.0 mmol/L (80-145 mg/dl) While we should attempt to treat people with diabetes to target, it is important to realise that failure to reach a target should not be seen as failure to achieve a benefit for the patient. All improvements are beneficial, whether or not a target is reached. The gold standard for assessment of long-term glycaemic control is HbA1c which reflects the glycaemic control over the previous two to three months. HbA1c should be used as the prime determinant of success of glycaemic control and of the need to change therapy. Where red cell turnover is shortened, e.g. in patients with haemoglobinopathies such as thalassaemia, the HbA1c is unreliably low. The urine microalbumin:creatinine ratio should preferably be done on the first void urine in the morning. There is considerable intra-individual variation (biological variation) in urine microalbumin levels even in healthy adults. In healthy individuals, the coefficient of variation (CV = mean/SD x 100%) of microalbumin to creatinine ratio in early morning urines (first void urine) is 31% whereas the CV is 103% on random spot urine samples obtained from healthy individuals. In diabetics, the CV of microalbumin to creatinine ratio on early morning urine specimens is 39%. 24-hour urine microalbumin excretion rate has a CV of 70% in healthy individuals (Howey, et al., Clinical Chemistry 1987,33: 2034-2038). There is now a global initiative to standardise HbA1c using the reference material, calibrators and primary reference methods developed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). An International Diabetes Working Group (IDWG) has been established comprising representatives from the International Diabetes Federation (IDF), European Association for the Study of Diabetes (EASD), American Diabetes Association (ADA), IFCC and the National Glycohemoglobin Standardisation Program (NGSP; DCCT-aligned). The IDWG has recommended that the IFCC-HbA1c should be used worldwide but implementation of this may be after 2008. The current direct comparison method used in the NGSP standardisation (method used in Direct Control and Complications Trial) is not very specific. Existing methods for measuring HbA1c (DCCT-aligned) have CVs (measure of imprecision) of around 3-4%. If an assay has a CV of 3% we cannot differentiate between an HbA1c value of 7.0% and 7.9%! IFCC-HbA1c values will be true HbA1c values. Methods for measuring HbA1c which are calibrated using the IFCC calibration materials have better precision than the DCCT-aligned methods, with intra-assay and inter-assay CVs of less than 2.0%. This improved precision may make it possible to use HbA1c to diagnose diabetes. Since the IFCC-HbA1c values are 'true' values they will be lower than the DCCT-aligned HbA1c values. The relationship between NGSP-HbA1c and IFCC-HbA1c is given by the master equation below and a comparison of NGSP-HbA1c and IFCC-HbA1c values is given in Table 3. Master Equation: NGSP-HbA1c = 0.915(IFCC-HbA1c) + 2.15% r2= 0.998 Table 3. Comparison of NGSP-HbA1c and corresponding IFCC-HbA1c values NGSP %HbA1c IFCC %HbA1c 4 2.1 5 3.1 6 4.2 7 5.3 8 6.4 9 7.5 10 8.5 11 9.6 12 10.7 Since the IFCC-HbA1c values are lower than the NGSP-HbA1c values there could be some confusion when the IFCC-HbA1c values are introduced. As such, the IDWG has recommended that the IFCC-HbA1c values should be reported as mean blood glucose levels. Studies are currently underway to assess the robustness of the relationship between IFCC-HbA1c and mean blood glucose level and to assess the dispersion of mean blood glucose values around the regression line. I will give a more detailed account of the global standardization of HbA1c in a future issue of the newsletter.

Dengue

By Prof. Datin Dr. G. Duraisamy   Clinical Consultant (Haematology) 

Dengue and dengue hemorrhagic fever (DHF) are caused by one of four closely related, but antigenically distinct, virus serotypes (DEN-1, DEN-2, DEN-3, and DEN-4), of the genus Flavivirus. Infection with one of these serotypes does not provide cross-protective immunity, so persons living in a dengue-endemic area can have four dengue infections during their lifetime. The viruses that cause it are maintained in a cycle that involves humans and Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans.

Dengue is a notifiable disease. The incidence of clinically diagnosed DHF has increased this past year. The case fatality rate for DHF has been increasing this year (2005). Dengue Haemorrhagic Fever (DHF) has

• High continuous fever for 2 to 7 days
• Haemorrhagic manifestations include petichiae, epistaxis, GIT bleeds and a positive tourniquet test (Hess's test)
  Tourniquet Test inflate blood pressure cuff to a point midway between systolic and diastolic pressure for 5 minutes
  Positive test: 20 or more petichiae per/inch² (6.25 cm²)
  The tourniquet test assesses capillary fragility.
• Hepatomegaly may occur on day 3 to 4 of the fever, more in children (>90%) than in adults (60%)
• Circulatory disturbances (shock in severe cases) = Dengue Shock Syndrome (DSS) - on day 2 to 7 of fever.Period of shock is short but life threatening.

Plasma Leakage causing haemoconcentration with a rising haematocrit or packed cell volume (PCV) andthrombocytopenia hallmarks the plasma leakage and abnormal haemostasis. This usually occurs before fever subsides and before the shock syndrome. Positive Tourniquet test and petichiae are observed early in the febrile phase. Mild gum bleeding, epistaxis, bruising at the site of venepuncture are early signs of abnormal haemostasis.


Lab Findings:

• Full blood count - WBC, platelets, hematocrit or PCV
  In patients with dengue, do serial hematocrits / PCV for hemoconcentration. Leukocyte counts are often low, and the patient may even be neutropenic.
  
  Atypical Lymphocytes with basophilic cytoplasm is seen.
  
  Platelet levels should also be checked, may fall to usually below 100 x 10⁹/dl, remains low for 3-5 days in most cases. It increases to normal rapidly during convalescence. Reduction of platelets follows that of leucopenia. Giant platelets may be seen, suggests increased platelet turn over.
  
  In shock platelet count may fall below 50 x 10⁹/dl. Risk of bleeding occurs if there is prolonged shock. This may be due to increased peripheral consumption & destruction of platelets. Impaired platelet function has been shown (shortened platelet survival time).
  
  Patients with bleeding manifestations should have serial hematocrit and platelet levels checked at least daily until their temperature is normal for 1-2 days.
  
  If a bone marrow is done, (in the febrile phase), there is hypocellularity with arrest of the maturity of all cell lines, especially megakaryocytes. During convalescence there is a rapid recovery of all the cell lines.
• Liver function tests - check albumin levels
• Urine - check for microscopic hematuria
• Coagulation Defects - Prolonged aPTT (activated Partial Thromboplastin Time) and PT (Prothrombin Time) may be seen with bleeding. There may be DIC (Disseminated Intravascular Coagulation).
• Dengue-specific tests
• Serology
  IgM ELISA test for Dengue antibodies

A convalescent-phase sample may also be drawn to test for IgM antibody. This sample should be drawn between 6 and 21 days after symptom onset.

If the blood sample was taken the first five days after the onset of symptoms, a convalescent-phase sample to measure IgM antibody is needed between 6-30 days after the onset of symptoms.

Gribbles performs the PanBio Rapid Strip Dengue Test for IgM and IgG and picks up primary, (increased IgM antibodies) and secondary (increased IgG) dengue infection. The test only takes 25-30 minutes to perform and uses a drop of sera.


Risk Factors Reported for DHF

• Virus strain
• Pre-existing anti-dengue antibody
• Previous infection
• Maternal antibodies in infants
• Host genetics
• Age
• Higher risk in secondary dengue infection
• Higher risk in locations with two or more serotypes circulating, hyperendemic at high levels (hyperendemic transmission).

In a subsequent infection, the pre-existing heterologous antibodies form complexes with the new infecting virus serotype, but do not neutralize the new virus.

Antibody-dependent enhancement is the process in which certain strains of dengue virus, complexed with non-neutralizing antibodies, can enter a greater proportion of cells of the mononuclear lineage, thus increasing g virus production.

Infected monocytes probably release vasoactive mediators, resulting in increased vascular permeability and hemorrhagic manifestations that characterize DHF and DSS.

Hepatitis B

A Diagnostic and Prognostic Mainstay of Hepatitis B Infection
Hepatitis B is a common enough infection in this country and, with its potential sequelae of cirrhosis and hepatocellular carcinoma being well recognized, is a regular source of diagnostic requests for the laboratory.

It is the purpose of this paper to give a review of the current tests in use, their diagnostic value and their interpretation.

The diagnosis of Hepatitis B infection is essentially based on serological techniques, which look for either the antigens or the corresponding antibodies of the different constituents of the causative virus. A battery of tests have been developed which when viewed in tandem as well as in sequence allow not only diagnoses of present and past infection but also activity status, chronicity and transmissibility, and, by extension, potential for serious liver damage and possible carcinogenic outcome.

The cornerstone of this battery of tests is the Hepatitis B surface antigen, HBsAg, the very presence of which defines active hepatitis B infection, and which becomes detectable in the blood within four weeks of exposure.

In the normal course of events as the body mounts its immune response, the surface antigen will be destined to disappear from the blood within about four months since onset. Almost at the same time as this antigen disappears antibodies to it i.e. anti-HBs will begin to appear. Detection of the antigen and its corresponding antibody thus have an inverse relationship vis a vis the determination of activity status as well as immunity to subsequent infections. In most instances these are mutually exclusive although there are some circumstances in which they may co-exist. In short, absence of HBsAg with presence of anti-HBs indicates recovery and immunity from future infections, a picture also seen as a successful response to immunization.

In a proportion of cases, however, the surface antigen will not disappear in the expected four months and if this persists beyond six months it is deemed that the infection has progressed to a chronic state, the disease is still active with potential for lasting liver damage and that the patient is able to continuously transmit the disease to others and hence become a chronic carrier.

It is at this stage that the other tests in the battery of serological tests become useful as prognostic and diagnostic tools. The Hepatitis Be antigen and its antibody anti-HBe are particularly useful in this regard and generally they too have an inverse relationship. Meaning that, detection of the e antigen in hepatitis B surface Ag positive case connotes high disease activity status with the potential for high transmissibility whilst an absence of the e antigen but anti-HBe positive points to the converse, although both of these may coexist for a short period in the early stage of the acute infection.

Accordingly, if the HBsAg is persistently positive, the detection of HBe is not a good sign. While its absence especially if anti-HBe is present is comforting. However there is a circumstance in which HBe may be absent in the face of high viral activity and that is a condition called a pre core mutation where there is a genetic mutation that renders the virus incapable of producing the e antigen although it is actively replicating. (The arbiter under these circumstances that will indicate the true picture of viral replication is the Hepatitis B virus DNA test which will be positive).

Another component of the virus that can throw light on the activity status of the infection is the core antigen HBc and its antibody anti-HBc. Ongoing replication of the virus throws up large amounts of core antigen but this is only found in the liver and not in the blood and hence not useful in serology tests. However the antibody anti-HBc as it emerges can be detected in the blood and the IgM form is found during the acute infection and lasts for about 6 months whilst the IgG form appears later in the disease but persists for life. Hence it is the IgM anti-HBc which is a useful marker of acute infection. Unlike anti-HBs, anti-HBc is not seen as a response to immunization.

A test being requested with increasing frequency is the PCR (Polymerase Chain Reaction) test for hepatitis B virus DNA. It is mainly used to provide a quantitative estimate of viral load, evidence of viral replication, progress of the disease and response to therapy. In general a high count is reflective of increased viral replication and thus disease activity. Response to therapy is indicated by significant drops in levels.

Therapy with interferon or lavimudine is recommended for cases in which chronic hepatitis is associated with the presence of e antigen, or if there is evidence of viral replication through a DNA test. When to stop therapy once initiated is also important as premature cessation can lead to relapse. The measure of viral load through the PCR test is useful in making this decision.

	HBsAg	Anti-HBs	Anti-HBc (IgM)	Anti-HBc (IgG)	HBe antigen	Anti-HBe	HBV-DNA PCR
Early infection	+						++
Recent infection			+	+	+		++
Immune		+		+		+	neg
Successful immunization		+					neg
Chronic active	+			+	+		+++
Chronic inactive	+			+		+	+
Indications for treatment	+			+	+		+++
Response to treatment	+			+	-	+	Dropping values +(-)
Relapse incomplete treatment	+			+	+/-		Rising values
Occult				+			+
HBV infection				+			+

                          *HBcAg - No blood test available (Histological marker)

Investigations for Chronic Hepatitis B

• HBsAg
• HBeAg, Anti-HBe
• HBV DNA (requires 4 ml of blood in EDTA or minimum 1.5ml of frozen plasma to be sent)
• LFT
• α fetoprotein
• Ultra sound liver

Treatment
- s/c α Interferon (IF) - 3 times a week or PEG ylated IF - S/C Weekly


Lamivudin
Problem of increasing resistance of HBV after 1 year.

Bacterial culture media

Bacterial culture media

Introduction

There are various reasons why bacteria have to be grown (cultured) in the laboratory on artificial culture media. One of the most important reasons being its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Indeed, isolating an organism from the clinical specimen is the first step in proving its role as an etiologic agent. Culturing bacteria is also the initial step in studying its morphology and its identification. Bacteria have to be cultured in order to obtain antigens from developing serological assays or vaccines. Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Culturing bacteria also provide a reliable way estimating their numbers (viable count). Culturing on solid media is another convenient way of separating bacteria in mixtures.

Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. Besides these, optimum pH, oxygen tension and osmolarity too have to be taken into consideration.

Ingredients
Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. While tap water is suitable for culture media, it must not be used if it contains high amount of minerals. In such situations, distilled or demineralised water should be used. Peptone is a byproduct of protein (plant or animal) digestion. Proteins are often obtained from heart muscle, casein, fibrin or soya flour and is digested using proteolytic enzymes such as pepsin, trypsin or papain. The final product contains peptones, proteoses and amino acids besides a variety of inorganic salts including phosphates, potassium and magnesium. Casein hydrolysate is obtained from hydrolysis of milk protein casein using HCl or trypsin. Meat extract is obtained by hot water extraction of lean beef and then concentrated by evaporation. Meat extract contains gelatin, albumoses, peptrones, proteoses, amino acids, creatinine, purines, and accessory growth factors. Yeast extract is prepared from washed cells of bakers’ yeast and contains wide range of amino acids, growth factors and inorganic salts. Malt extract is prepared by extracting soluble materials from sprouted barley in water at 55oC and concentrated by evaporation. It contains maltose, starch, dextrin, glucose and small amounts of protein and protein breakdown products and growth factors.

Brief history
Initially, culture media were very simple; Louis Pasteur used simple broths made up of urine or meat extracts. Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (35-37oC) and was digested by certain bacteria.

Classification
Bacterial culture media can be classified in at least three ways; Based on consistency, based on nutritional component and based on its functional use.
Classification based on consistency:
Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as “broths” (e.g nutrient broth).
Liquid media are available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods).

Solid media:
Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolysed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders.

For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at ~50oC, it is poured on to sterile Petri plates and allowed to set. For preparing agar containing media in test-tubes, the culture medium is mixed with 3% agar and heated with stirring to melt. This ensures that all the tubes get equal amounts of agar. These tubes can then be sterilized by autoclaving.

Semi-solid media
Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragie’s tube). Certain transport media such as Stuart’s and Amies media are semi-solid in consistency. Hugh & Leifson’s oxidation fermentation test medium as well as mannitol motility medium are also semi-solid.

Biphasic media
Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture.
Biphasic medium

Other solidifying agents
Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum containing medium such as Loeffler’s serum slope and egg containing media such as Lowenstein Jensen medium and Dorset egg medium are solidified as well as disinfected by a process of inspissation.

Classification based on nutritional component:
Media can be classified as simple, complex and synthetic (or defined). While most of the nutritional components are constant across various media, some bacteria need extra nutrients. Those bacteria that are able to grow with minimal requirements are said to non-fastidious and those that require extra nutrients are said to be fastidious. Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar have ingredients whose exact components are difficult to estimate. Synthetic or defined media such as Davis & Mingioli medium are specially prepared media for research purposes where the composition of every component is well known.

Classification based on functional use or application:
These include basal media, enriched media, selective/enrichment media, indicator/differential media, transport media and holding media.
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basal medium
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media.
Blood agar is preparing by adding 5-10% (by volume) to a basal medium such as nutrient agar or other blood agar bases. Since blood can not be sterilized, it has to be collected aseptically from the animal. Animals have to be bled and the blood is collected in sterile containers with anticoagulant or glass beads. While sheep blood is preferred, blood from rabbit, horse and ox can also be collected. Human blood must be avoided since it may contain inhibitory substances including antibiotics. After the blood agar base is autoclaved, blood is added to the medium at temperature just above the solidifying point of agar. The mixture is then poured on to the plates and allowed to solidify. Blood agar is useful in demonstrating hemolytic properties of certain bacteria. Two major types of hemolysis are often seen on blood agar; beta and alpha hemolysis. Beta hemolysis is the complete lysis of RBC resulting in clearing around the colonies whereas alpha hemolysis is the partial lysis of RBC resulting in greenish discolouration around the colonies. Gamma hemolysis is a misnomer and it indicates non-hemolytic colonies. Chocolate agar is also known as heated blood agar or lysed blood agar. The procedure is similar to that of blood agar preparation except that the blood is added while the molten blood agar base is still hot. This lyses the blood cells and releases their contents into the medium. This process turns the medium brown, hence the name. This medium is especially useful in growing Hemophilus and Neisseria.
Serum for medium can be obtained from animal blood but must be filtered through membrane or seitz filter before use.

Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% Potassium tellurite. McConkey’s Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide. Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating Malachite green. Wilson & Blair’s Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green. Selective media such as TCBS Agar and Monsur’s Tellurite Taurocholate Gelatin Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6), which inhibits most other bacteria.

Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens.

Differential/Indicator media:
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. When a particular carbohydrate is incorporated into a medium and a mixture of bacteria inoculated on it, only that bacterium that can ferment it produces acid. This change in pH is detected by using a pH indicator incorporated in the medium and the bacterium that can ferment the sugar appears in a different colour. This approach is used in MacConkey’s agar, CLED agar, TCBS agar, XLD agar etc. MacConkey’s agar is the most commonly used media to culture and identify gram negative bacilli (especially enterobacteriaceae members). It contains bile salts (selective agent), lactose (sugar), peptone and neutral red (pH indicator), agar and water. Those bacteria that can ferment lactose produce pink coloured colonies where non-lactose fermenting colonies produce colourless colonies. Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose containing TCBS medium.
Reduction of potassium tellurite to metallic tellurium by Corynebacterium diphtheriae results in production of black coloured colonies on PT agar. Production of H2S by Salmonella typhi results in production of black coloured colonies on Wilson & Blair’s medium. Enterococcus fecalis produces black coloured colonies on bile esculin agar due to reduction of esculin to esculetin. Detection of hemolysis on blood agar can be considered as an indicator property of Blood agar.

Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium are used to transport feces from suspected cholera patients. Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery. Pike’s medium is used to transport streptococci from throat specimens.

Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Robertson cooked meat that is commonly used to grow Clostridium spps medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colourless.

Preparation and storage:
Care must be taken to adjust the pH of the medium before autoclaving. Various pH indicators that are in use include phenol red, neutral red, bromothymol blue, bromocresol purple etc. Dehydrated media are commercially available and must be reconstituted as per manufacturers’ recommendation. Most culture media are sterililized by autoclaving. Certain media that contain heat labile components like glucose, antibiotics, urea, serum, blood are not autoclaved. These components are filtered and may be added separately after the medium is autoclaved. Certain highly selective media such as Wilson and Blair’s medium and TCBS agar need not be sterilized. It is imperative that a representation from each lot be tested for performance and contamination before use. Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room temperature for weeks.

Gram stain

Gram stain

The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. Gram staining is not used to classify archaea, since these microorganisms give very variable responses.

Gram staining consists of four components:
Primary stain (Crystal violet, methyl violet or Gentian violet)
Mordant (Gram's Iodine)
Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)
Counterstain (Dilute carbol fuchsin, safranin or neutral red)

The original description of staining technique by Christian Gram in a publication titled "The differential staining of Schizomycetes in tissue sections and in dried preparations" in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189 was slightly different from what we use today. The primary stain used was aniline gentian violet, mordant was Lugol's iodine (iodine-potassium iodide in water), decolorizer was absolute alcohol and bismark brown was the counterstain.

Procedure:

The smear on a glass slide is covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide is washed in water.

The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant.

The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. A mixture of ecetone-ethyl alcohol (1:1) can also be used for decolorization. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolorization is the most crucial part of Gram staining and errors can occur here. Prolonged decolorization can lead to over-decolorized smear and a very short decolorization period may lead to under-decolorized smear.

After the smear is decolorized, it is washed in water without any delay. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat fixed before observing under microscope.

From bacteriology

Those bacteria that hold on to primary dye-iodine complex and remain violet are called Gram positive and those which get decolorized and subsequently take up counterstain (pink/red) are called Gram negative.

Basic fuchsin (present in dilute carbol fuchsin) stains many Gram negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin.

In order to ascertain if the staining procedure was satisfactorily conducted, a control smear of known Gram positive organism (e.g., Staphylococcus aureus) and a known gram negative organism (Escherichia coli) must be stained simultaneously. While the fibrin in a clinical specimen may appear gram positive, the pus cells and epithelial cells are always gram negative.

Mechanism of Gram reaction:

Various theories have been proposed to explain why some bacteria retain the dye and some don't. Theories such as differences in cytoplasmic pH (2 in case of Gram positive bacteria and 3 in case of Gram negative bacteria), and presence of Magnesium ribonucleate in Gram positive bacteria and its absence in Gram negative bacteria have not received widespread acceptance. The thickness of Gram positive cell wall and presence of more lipids in Gram negative cell walls have been more acceptable reasons for Gram stain reactions.

It is believed that the positively charged crystal violet pass through the cell wall and cell membrane and binds to negatively charged components inside the cell. Addition of negatively charged iodine (in the mordant) binds to the positively charged dye and forms a large dye-iodine complex within the cell. Crystal violet (hexamethyl-para-rosaniline chloride) interacts with aqueous KI-I2 via a simple anion exchange to produce a chemical precipitate. The small chloride anion is replaced by the bulkier iodide, and the complex thus formed becomes insoluble in water. During decolorization, alcohol dissolves the lipid present in the outer membrane of Gram negative bacteria and it leaches the dye-iodine complex out of the cell. A thin layer of peptidoglycan does not offer much resistance either. The dye-iodine complexes are washed from the Gram negative cell along with the outer membrane. Hence Gram negative cells readily get decolorized. On the other hand Gram positive cells become dehydrated from the ethanol treatment, closing the pores as the cell wall shrinks during dehydration. The dye-iodine complex gets trapped inside the thick peptidoglycan layer and does not get decolorized.

Limitations of Gram staining:

Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram positive bacteria may appear pink and when under-decolorized gram negative bacteria may appear Gram positive.

The Gram reaction also depends on the age of the cell. Old cultures of Gram positive bacteria (where cell walls may be weakened) may readily get decolorized. Gram positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram negative. Gram-positive bacteria such Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these cells. In cultures of Bacillus, and Clostridium a decrease in peptidoglycan thickness during cell growth may cause some of them to appear Gram negative.

Certain group of bacteria can display variable response to the stain, which can be due to growth stress (e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that results in a number of nonviable, gram-negative cells in a gram positive culture, but certain bacterial species are known for their gram variability even under optimal growth conditions. Some bacteria tend to appear Gram negative when grown in acidic medium.

Loss of cell walls in Gram positive bacteria may render them Gram negative (L-forms). Bacteria totally devoid of cell wall (Mycoplasma) are always Gram negative. Bacteria such as Mycobacterium that have extra waxy content in their cell wall are difficult to stain. Small and slender bacteria such as Treponema, Chlamydia, Rickettsia are often difficult to stain by Gram's method. Gram positive bacteria that have been phagocytosed by polymorphs may also appear Gram negative.

Modifications of Gram stain:

There have been several modifications of Gram's stain. These are:
1. Kopeloff and Beerman's modification: Primary stain solution consists of freshly constituted methyl violet with sodium bicarbonate in distilled water. Mordant consists of iodine dissolved in 4% NaOH solution. Decolorization is either using acetone alone or a mixture of acetone and ethanol. Basic fuchsin is used to counterstain the smear. This method may be modified to stain tissue sections.

2. Jensen's modification: This method involves use to methyl violet as primary stain, iodine and potassium iodide in water as mordant, absolute alcohol as decolorizer and neutral red as counterstain. For Neisseria spp, Sandiford's counterstain is useful.

3. Weigert's modification: This modification is particularly useful for staining tissue sections. The primary stain carbol gentian violet is prepared using saturate alcoholic solution of gentian violet and 5% phenol solution. Gram's iodine is used as a mordant and aniline-xylol is used as a decolorizer. The counterstain carmalum (carminic acid and potassium alum in water), however is used ahead of primary stain. This method may be used to stain Pneumocystis cysts.

4) Preston and Morrell's modification: The primary stain used in this modification is ammonium oxalate-crystal violet. The smear is washed in Lugol's iodine and further treated with iodine solution. The smear is decolorized using iodine-acetone decolorizer and counterstained using dilute carbol fuchsin solution. This method has been further modified to overcome the irritating iodine in aerosols by reducing the iodine concentration to one-tenth and shortening the duration of decolorization to ten seconds.

Applications of Gram staining:

Differentiation of bacteria into Gram positive and Gram negative is the first step towards classification of bacteria.
It also the first step towards identification of bacteria in cultures.
Observation of bacteria in clinical specimens provides a vital clue in the diagnosis of infectious diseases.
Useful in estimation of total count of bacteria.
Empirical choice of antibiotics can be made on the basis of Gram stain’s report.
Choice of culture media for inoculation can be made empirically based on Gram’s stain report.

Miscellanea: 

Although Gram stain is useful in staining bacteria, certain fungi such as Candida and Cryptococcus are observed as Gram positive yeasts.
Half-Gram stain refers to modified staining technique, where the smear is neither decolorized nor counterstained. It is useful to stain a known Gram positive bacterium.
Rapid Gram stain refers to quickened technique where the smear is exposed to only 30 seconds instead of one minute.
In specimen such as sputum, capsulated bacteria may stand out as clear spaces between the bacterium and the pink (mucus) background.
The spores may stand out as clear, unstained region in sporing bacteria.

Tables of Serum Chemical Constituents

Tables of Serum Chemical Constituents
Small (non-protein) species

Serum Analyte	Reference Ranges	Increased In	Decreased In
Acetone	<1 mg/dL	Diabetic ketoacidosis, Starvation, Glycogen storage disease, Alcoholic intoxication	NA
Acetoacetate	<1 mg/dL	Diabetic ketoacidosis, Starvation, Glycogen storage disease, Alcoholic intoxication	NA
Ammonia	Adult: 15 – 110 ug/dLNeonate: 90 – 150 ug/dL	Hepatitis, Cirrhosis, Reye syndrome, GI bleeding/obstruction	Malignant hypertension
Bilirubin, direct(conjugated)	Adult/Child: 0.1 – 0.3 mg/dL	Jaundice, Biliary obstruction, Drug cholestasis	NA
Bilirubin, indirect(unconjugated)	Adult/Child: 0.2 – 0.8 mg/dLNeonate: 1- 12 mg/dL	Hemolytic jaundice, HDN, Hepatitis, Cirrhosis, Sepsis, Pernicious anemia, Sickle cell anemia, Transfusion reaction	NA
Bilirubin, total	Adult/Child: 0.2 – 1.0 mg/dL Neonate: 1.9 - 12 mg/dLCritical: >15.0 mg/dL	Hemolytic jaundice, HDN, Hepatitis, Cirrhosis, Sepsis, Pernicious anemia, Sickle cell anemia, Transfusion reaction	NA
Calcium, ionized	Adult: 4.5 – 5.6 mg/dLNeonate: 4.20 – 5.58 mg/dL	Hyperparathyroidism, renal/lung carcinoma-producing PTH, Paget’s disease, Vitamin D intoxication, Addison’s disease, Acromegaly	Hypoparathyroidism, Renal failure, Rickets, Vitamin D deficiency, Osteomalacia, Alkalosis, Pancreatitis.
Calcium, total	Adult: 9.0 – 10.5 mg/dL Neonate: 9.0 – 10.6 mg/dLCritical: <6.0 or >14.0 mEq/L	Hyperparathyroidism, renal/lung carcinoma-producing PTH, Paget’s disease, Vitamin D intoxication, Addison’s disease, Acromegaly	Hypoparathyroidism, Renal failure, Rickets, Vitamin D deficiency, Osteomalacia, Alkalosis, Pancreatitis. Critical: <6.0
Carbon Dioxide Content(CO2 Content)	Adult: 23 – 30 mEq/L Infant: 20 – 28 mEq/L Neonate: 13 – 22 mEq/L (as bicarbonate, HCO3-)Critical: <6.0 mEq/L	Severe diarrhea, Starvation, Severe vomiting, Aldosteronisn, Emphysema, Metabolic alkalosis	Renal failure, Salicylate toxicity, Diabetic ketoacidosis, Metabolic acidosis, Shock
Chloride	Adult/Child: 98 – 106 mEq/LCritical: <80 or >115 mEq/L	Dehydration, Cushing’s syndrome, Metabolic acidosis, Hyperventilation, Respiratory alkalosis, Renal dysfunction	Overhydration, CHF, Vomiting, Respiratory acidosis, Addison’s disease, Metabolic alkalosis, Aldosteronism, Burns
Cholesterol, total	Adult/Child: 120 – 200 mg/dL Infant: 70 – 175 mg/dLNeonate: 53 – 135 mg/dL	Hypothyroidism, Diabets Mellitus, Nephrotic syndrome, Hypertension, Atherosclerosis, MI, Nephrosis, High-cholesterol diet, Nephrosis	Malabsorption, Sepsis, Malnutrition, Liver disease, AMI
Creatinine	Adult/Child: 0.5 – 1.2 mg/dL	Nephritis, Renal necrosis, Diabetic nephropathy, CHF, Atherosclerosis, Acromegaly, Shock	Debilitation, Decreased muscle mass (Muscular systrophy, Myasthenia gravis)
Glucose, fasting	Adult/Child: 65 – 110 mg/dL Neonate: 30 – 60 mg/dLCritical: <40 or >400 mg/dL	Diabetes mellitus, Cushing’s syndrome, Acute pancreatitis, Corticosteroid therapy, Acromegaly	Insulin overdose, Hypothyroidism, Hypopituitarism, Addison’s disease, liver disease, starvation
Iron	Adult/Child: 50 – 175 ug/dLNeonate: 100 – 250 ug/dL	Hemosiderosis, Hemochromatosis, Hemolytic anemia, Hepatitis, Hepatic necrosis, Iron poisoning	Inadequate dietary iron, Chronic blood loss, Iron deficiency anemia, Inadequate iron absorption
Lactate	5-12 mg/dL	Shock, Tissue ischemia, Severe liver disease, Carbon monoxide poisoning	NA
Lead (whole blood)	Adult: <40 ug/dL Child: <25 ug/dL Critical: >40 ug/dL Toxic: >100 ub/dL	-	-
Magnesium	Adult: 1.2 – 2.1 mEq/L Child: 1.4 – 1.8 mEq/L Neonate: 1.2 – 1.8 mEq/LCritical: <0.5 or >3.0 mEq/L	Renal insufficiency, Uncontrolled diabetes, Addison’s disease, Hypothyroidism, Magnesium antacid ingestion	Malnutrition, Malabsorption, Hypoparathyroidism, Alcoholism, Diabetic acidosis
Phosphorus, inorganic(as phosphate)	Adult: 3.4 – 4.5 mg/dL Child: 4.5 – 6.5 mg/dL Neonate: 4.3 – 9.3 mg/dLCritical: <1.0 mg/sL	Renal failure, Increased intake, Acromegaly, Hypoparathyroidism, Bone metastisis, Sarcoidosis, Hypocalcemia, Liver disease, Acidosis	Malnutrition, Sepsis, Hyperparathyroidism, Hypercalcemia, Alkalosis, Alcoholism, Vitamin D deficiency, Rickets, Sepsis, Alkalosis
Potassium	Adult: 3.5 – 5.0 mEq/L Neonate: 3.9 – 5.9 mEq/LCritical: <2.5 or >6.5 mEq/L (Neonate: <2.5 or >8.0 mEq/L)	Excessive intake, Renal failure, Acidosis, Hypoaldosteronism, Hemolysis, Dehydration, Tissue crush injury	Deficient intake, Burns, Diarrhea or vomiting, Diuresis, Cushing’s syndrome, Licorice ingestion, Ascites, Cystic fibrosis
Sodium	Adult/Child: 136 – 145 mEq/L Neonate: 134 – 144 mEq/LCritical: <120 or >160 mEq/L	Increased intake, Cushing’s syndrome, Hyperaldosteronism, Profound sweating, Diabetes insipidus	Decreased intake, Ascites, Addison’s disease, CHF, Diuresis, Diarrhea or vomiting, Edema, Pleural effusion
Thyroid Uptake(TU, T3-Uptake)	25 – 35%	Interpretation is dependent on Thyroxine and FTI values	Interpretation is dependent on Thyroxine and FTI values
Thyroxine, free(free T4)	Adult: 0.8 – 2.7 ng/dL Child: 0.8 – 2.0 ng/dLNeonate: 2.0 – 6.0 ng/dL	Grave’s disease, Plummer’s disease, Toxic thyroid adenoma, Acute thyroiditis, Hyperthyroidism	Hypothyroidism, Myxedema, Pituitary insufficiency, Cirrhosis, Hypothalmic failure, Renal failure, Cushing’s syndrome, Liver diseases
Thyroxine, total(total T4)	Adult: 4.0 – 12.0 ug/dL Child: 5.0 – 15.0 ug/dL Neonate: 10.0 – 22.0 ug/dLCritical: <2.0 or >20.0 ug/dL	Grave’s disease, Plummer’s disease, Toxic thyroid adenoma, Acute thyroiditis, Hyperthyroidism	Hypothyroidism, Myxedema, Pituitary insufficiency, Cirrhosis, Hypothalmic failure, Renal failure, Cushing’s syndrome, Liver diseases
Triglycerides	Adult Male: 40 – 160 mg/dL Adult Female: 35 – 135 mg/dL Child: 30 – 163 mg/dL(age/sex dependent)	Glycogen storage disease, Hyperlipidemia, Diabetes mellitus, CHD, Nephrotic syndrome, Hypertension, Cirrhosis, Pregnancy, MI	Malabsorption, Malnutrition, Hyperthyroidism
Triiodothyronine (T3)(T3 RIA)	Adult: 40 – 205 ng/dL Child: 80 – 270 ng/dLNeonate: 100 – 740 ng/dL	Grave’s disease, Plummer’s disease, Toxic thyroid adenoma, Acute thyroiditis, Hyperthyroidism	Hypothyroidism, Myxedema, Pituitary insufficiency, Cirrhosis, Hypothalmic failure, Renal failure, Cushing’s syndrome, Liver diseases
Urea Nitrogen (BUN)	Adult: 8.0 – 22 mg/dL Child: 5.0 – 18 mg/dLNeonate: 3.0 – 12.0 mg/dL	Hypovolemia, Shock, Burns, Dehydration, CHF, MI, High protein intake, Starvation, Sepsis, Renal disease, Renal failure, Ureteral obstruction	Liver failure, Malnutrition, Malabsorption, Nephrotic syndrome
Uric Acid	Adult Male: 2.1 – 8.5 mg/dL Adult Female: 2.0 – 6.6 mg/dLChild: 2.5 – 5.5 mg/dL	Gout, Multiple myeloma, Leukemias, Renal disease, Acidosis, Toxemia of pregnancy, Alcoholism, Shock, Hypothyroidism, High purine diet	Wilson’s disease, Fanconi syndrome, Lead poisioning, Yellow liver atrophy

Enzymes

Serum Analyte	Reference Ranges	Increased In	Decreased In
Alanine aminotransferase(ALT, SGPT)	Adult/Child: 5 – 35 IU/L	Hepatitis, Cirrhosis, Hepatic tumor, Hepatotoxic drugs, Obstructive jaundice, MI, Muscle trauma, Myositis, Infectious Mononucleosis	NA
Aldolase	Adult: 3.0 - 8.2 SL U/dl	Hepatitis, Muscular dystrophy, MI, polymyositis, Muscle injuries	Late Muscular dystrophy, Muscle-wasting disease, Fructose intolerance
Acid phosphatase,prostatic	Adult male: 0.2 – 3.5 U/LAdult female: 0.0 – 0.8 U/L	Prostatic carcinoma, Multiple myeloma, Prostate manipulation, Prostatitis, Cancer of breast or bone, Cirrhosis, Hyperparathyroidism, Renal impairment	NA
Acid phosphatase, total	Adult male: 2.5 – 11.7 U/LAdult female: 0.3 – 9.2 U/L	Prostatic carcinoma, Multiple myeloma, Prostate manipulation, Prostatitis, Cancer of breast or bone, Cirrhosis, Hyperparathyroidism, Renal impairment	NA
Alkaline phosphatase, total	Adult: 30 – 120 U/L Child/adolescent: <2yr: 85 – 235 U/L 2 – 8 yr: 65 – 210 U/L 9 – 15 yr: 60 – 300 U/L16 – 21 yr: 30 – 200 U/L	Cirrhosis, Biliary obstruction, Hepatic tumor, 3rd Trimester pregnancy, Metastatic tumor to bone, Healing fracture, RA, Sarcoidosis	Hypothyroidism, Malnutrition, Pernicious anemia, Scurvy, Celiac diseade, High Vit. B intake
Amylase, alpha	30 – 220 U/L	Acute pancreatitis, peptic ulcer, Necrotic bowel, Acute cholecystitis, Mumps, Pulmonary infarction, Diabetic ketoacidosis, Duodenal obstruction	NA
Angiotensin-converting enzyme (ACE)	Adult: 23 – 57 U/mLChildren: much higher	Sarcoidosis, Gaucher’s disease, Tuberculosis, Leprosy, Cirrhosis, Histoplasmosis, Hodgkin's disease, Myeloma, Pulmonary fibrosis, Scleroderma, Amyloidosis, Hyperthyroidism, <20 yr old	NA
Aspartate aminotransferase(AST, SGOT)	Adult: 0 – 35 U/LNewborn: 15 – 60 U/L	MI, Hepatitis, Cirrhosis, Drug-induced liver injury, Hepatic metastasis/necrosis, Infectious mononucleosis, Muscle trauma/diseases, Acute pancreatitis	Acute renal disease, Diabetic ketoacidosis, Pregnancy, Renal dialysis
Cholinesterase, pseudo-(Pseudocholinesterase)	5 – 15 mg/L7 – 19 kU/L	Reticulocytosis, Hyperlipidemia, Nephrosis, Diabetes	Organic phosphate insecticide poisoning, Hepatocellular disease, Congenital enzyme deficiency, Malnutrition, Drugs: atropine, caffeine, codeine, estrogens, morphine, neostigmine, phenothiazines, theophylline, quinidine, vitamin K
Creatine phosphokinase, total(CPK, CK)	Adult male: 55 – 170 U/L Adult female: 30 – 135 U/LNewborn: 68- 580 U/L	Cardiac muscle disease/injury, Skeletal muscle disease/injury, CNS (brain) disease/injury, Strenuous physical exercise, IM injections	NA
Creatine phospokinase, BB isoenzyme(CPK-BB, CPK1)	0.0 % of total CPK	CNS diseases, Adenocarcinoma of breast or lung, Pulmonary infarction	NA
Creatine phosphokinase, MB isoenzyme(CPK-MB, CPK2)	0.0 % of total CPK	AMI, Cardiac aneurysm surgery, Cardiac ischemia, Cardiac defibrillation, Myocarditis, Ventricular arrhythmias	NA
Creatine phosphokinase, MM isoenzyme(CPK-MM, CPK3)	100 % of total CPK	Rhabdomyolysis, Muscular dystrophy, Myositis, IM injections, Muscle injury, convulsions, Hypokalemia, Hypothyroidism	NA
Gamma-glutamyl transpeptidase(GGTP, g-GTP, g-GT)	Adult male, female >45 yr: 8 – 38 U/L Adult female <45 yr: 5 – 27 U/L Child: same as adultNewborn: 40 – 190 U/L	Hepatitis, Cirrhosis, Hepatic necrosis, Hepatic carcinoma, Hepatotoxic drugs, Cholestasis, MI, Pancreatitis, Pancreatic carcinoma, Infectious Mononucleosis, CMV, Reye’s syndrome	NA
Lactate dehydrogenase, total(LDH, LD)	0 – 4 day: 290 – 775 U/L 4 – 10 day: 545 – 2000 U/L 10d – 2 yr: 180 – 430 U/L 2 – 12 yr: 110 – 295 U/L 12 – 60 yr: 100 – 190 U/L> 60 yr: 110 – 210 U/L	MI, Pulmonary disease, Hepatic disease, Anemias, Muscle disease or injury, Renal parenchymal disease, Intestinal ischemia, Testicular carcinoma, Lymphoma, Advanced carcinoma, Pancreatitis, Hemolysis	Ascorbic acid
Lactate dehydrogenase, isoenzymes(LDH-1, LDH-2, LDH-3, LDH-4, LDH-5)	LDH-1: 17 – 27 % of total LDH-2: 27 – 37 % of total LDH-3: 18 – 25 % of total LDH-4: 3 – 8 % of totalLDH-5: 0 – 5 % of total	LDH-1: Cardiac LDH-2: RE system LDH-3: Lung and other tissues LDH-4: Kidney, Placenta, PancreasLDH-5: Liver, Muscle	Ascorbic acid
Leucine aminopeptidase(LAP, arylamidase)	Adult male: 80 – 200 U/mLAdult female: 75 – 185 U/mL	Hepatitis, Cirrhosis, Hepatic ischemia, Hepatic necrosis, Hepatic carcinoma, Hepatotoxic drugs, Cholestasis, Gallstones	NA
Lipase	0 – 417 U/L	Acute pancreatitis, Chronic pancreatitis, Pancreatic carcinoma, Acute cholecystitis, Cholangitis, Extrahepatic duct obstruction, Renal failure, Bowel obstruction, Salivary gland inflammation or tumor, Peptic ulcer	NA
Renin (PRA)	Adult (upright, sodium-restricted diet) <40 yr: 2.9 – 24.0 ng/mL/hr >40 yr: 2.9 – 10.8 ng/mL/hr Adult (upright, sodium-replete diet) <40 yr: 0.1 – 4.3 ng/mL/hr >40 yr: 0.1 – 3.0 ng/dL/hr	Hypertension, Chronic renal failure, Salt-losing GI disease (vomiting/dirrhea), Addison’s disease, Renin-producing renal tumor, Cirrhosis, Hyperkalemia, Hemorrhage	Primary hyperaldosteronism, Steroid therapy, Congenital adrenal hyperplasia

Serum or Plasma Hormones, Hormone Precursors and Derivatives

Hormone	Ref. Intervals	Constitution	Source/Target	Function
Thyrotropin-releasing hormone (TRH)	Baseline TSH: <10 mU/mLStimulated TSH (following iv TRH): 2´baseline	Peptide (3 aa)*	H/Anterior pituitary lobe	Release of TSH and PRL
Gonadotropin-releasing hormone (GnRH) or luteinizing hormone- releasing hormone (LHRH)	-	Peptide (10 aa)	H/Anterior pituitary lobe	Release of LH and FSH
Corticotropin-releasing hormone (CRH)	-	Polypeptide (41 aa)	H/Anterior pituitary lobe	Release of ACTH and ß-LPH
Growth hormone-releasing hormone (GHRH)	-	Polypeptide (40 aa)	H/Anterior pituitary lobe	Release of GH
Somatostatin± (SS) or growth hormone-inhibiting hormone (GHIH)	-	Peptide (14 aa)	H/Anterior pituitary lobe	Suppression of GH and TSH; inhibition of gastrin, VIP, GIP, secretin, motilin, and insulin
Prolactin-releasing factors (PRF)	-	Peptide?	H/Anterior pituitary lobe	Release of PRL
Prolactin-inhibiting factor (PIF)	0 – 20 pg/mL(as dopamine)	Dopamine	H/Anterior pituitary lobe	Suppression of PRL
Thyrotropin or thyroid-stimulating hormone (TSH)	40 – 200 mg/dL	Glycoprotein‡ (a , 89 aa; ß, 112 aa)	AP/Thyroid gland	Stimulation of thyroid hormone formation and secretion
Follicle-stimulating hormone (FSH)	Male: (<45 yr): 4 – 25 IU/L (³ 45 yr): 2 – 14 IU/L Female: (Non-preg, 18 – 40 yr): 4 – 30 IU/L (Midcycle): 10 – 90 IU/L(Postmenopausal): 40 – 250 IU/L	Glycoprotein‡ (a , 89 aa; ß, 115 aa)	AP/Ovary AP/Testis	Growth of follicles and, with LH, secretion of estrogens and ovulation. Development of seminiferous tubules, spermatogenesis
Luteinizing hormone (LH)	Female: (Non-preg, 18 – 40 yr): 1 – 9 IU/L (Midcycle): >11 IU/L (Postmenopausal): 13 – 60 IU/LMale: 6 – 23 IU/L	Glycoprotein‡ (a , 89 aa; ß, 115 aa)	AP/Ovary AP/Testis	Ovulation, formation of corpora lutea, secretion of progesterone. Stimulation of interstitial tissue; secretion of androgens.
Prolactin (PRL)	Female: 80 – 530 mIU/LMale: 80 – 350 mIU/L	Protein (198 aa)	AP/Mammary gland	Proliferation of mammary gland; initiation of milk secretion; antagonist of insulin action
Growth hormone (GH) or somatotropin	Female: 0 – 8.0 m g/LMale: 0 – 4.0 m g/L	Protein (191 aa)	AP/Body as a whole	Growth of bone and muscle
ß-Lipotropin (ß-LPH)	-	Polypeptide (91 aa)	AP/Unknown	Precursor of ß-MSH and the endorphins
Corticotropin or adrenocorticotropin (ACTH)	1.2 – 15.6 pmol/L(ng/L = 4.5 ´ pmol/L)	Polypeptide (39 aa)	AP/Adrenal cortex	Stimulation of adrenocortical steroid formation and secretion
ß-Endorphin (ß-END)±¶	5 – 35 pmol/L	Polypeptide (31 aa)	AP/Brain	Endogenous opiate; raising of pain threshold and influence on extrapyramidal motor activity
a -Melanocyte-stimulating hormone (a-MSH)	-	Peptide (13 aa)	AP/Skin	Dispersion of pigment granules, darkening of skin
Leu-enkephalin (LEK)±¶ and met-enkephalin (MEK)±¶	-	Peptide (5 aa)	AP/Brain	Endogenous opiate; raising of pain threshold and influence on extrapyramidal motor activity
Vasopressin or antidiuretic hormone (ADH)	0.0 – 7.0 pmol/L	Peptide (9 aa)	PP/Arterioles PP/Renal tubules	Elevation of blood pressure.Water reabsorption
Oxytocin	<3.2 m IU/mLOxytocin challenge test: Negative	Peptide (9 aa)	PP/Smooth muscle (uterus, mammary gland)	Contraction, action in parturition and in sperm transport, ejection of milk
Serotonin or 5-hydroxytryptamine (5-HI)	5 – 220 ng/mL	Indoleamine	PG/Cardiovascular, respiratory, and gastrointestinal systems, brain	Neurotransmitter; stimulation or inhibition of various smooth muscles and nerves; possible role in mental illness
Melatonin	-	Indoleamine	PG/Hypothalamus	Suppression of gonadotropin and GH secretion; induction of sleep
Thyroxine (T4) and triiodothyronine (T3)(free T4, free T3)	T4: (<1 m): 0.8 – 2.2 ng/dL (1-6 m): 0.8 - 1.8 ng/dL (6m-1y): 0.8 – 1.6 ng/dL (1-12y): 0.9 – 1.4 ng/dL (>12 yr): 0.8 – 1.5 ng/dLT3: 2.2 – 4.0 pg/mL	Iodoamino acids	TG/General body tissue	Stimulation of oxygen consumption and metabolic rate of tissue
Calcitonin or thyrocalcitonin	<15 pmol/L	Polypeptide (32 aa)	TG/Skeleton	Inhibition of calcium resorption; lowering of plasma calcium and phosphate
Parathyroid hormone (PTH) or parathormone	1.7 – 7.3 pmol/L	Polypeptide (84 aa)	PTG/Skeleton, kidney, gastrointestinal tract	Regulation of calcium and phosphorus metabolism
Cortisol	AM peak: 200 – 650 nmol/LPM trough: <50% peak	Steroid	AC/General body tissue	Metabolism of carbohydrates, proteins, and fats; inflammation, resistance to infection; hypersensitivity
Aldosterone	100 – 800 pmol/L	Steroid	AC/Kidney	Salt and water balance
Norepinephrine and epinephrine	Epinephrine (pg/mL) 2-10 d: 36-400 11d-3m: 55-200 4-11mo: 55-440 12-23m: 36-640 24-35m: 18-440 3-17yr: 18-460 18+ yr: 10-200 Norepinephrine (pg/mL) 2-10 d: 170-1180 11d-3m: 370–2080 4-11mo: 270–1120 12-23m: 68–1810 24-35m: 170–1470 3-17yr: 85–125018+ yr: 80–520	Aromatic amines	AM/Sympathetic receptors	Stimulation of sympathetic nervous system
Epinephrine	<570 pmol/L(see above)	Aromatic amine	AM/Liver and muscle, adipose tissue	GlycogenolysisLipolysis
Estrogens	Female estradiol (pmol/L): Early follicular: 100 – 200 Preovulatory: 500 – 1700 Luteal: 500 – 900 Postmenopausal: 70 – 200 Male: 0 – 283 pmol/LEstriol: Varies with gestational age (increases thru pregnancy)	Phenolic steroids	O/Female accessory sex organs	Development of secondary sex characteristics
Progesterone	Female: (nmol/L) Follicular: 2.0 – 4.5Luteal: 7.0 – 70.0	Stroid	O/Female accessory reproductive structure	Preparation of the uterus for ovum implantation, maintenance of pregnancy
Relaxin	-	Polypeptide	O/Uterus	Inhibition of myometrial contraction
Inhibin	Female (>16 yr):0 – 78 pg/mL	Polypeptide	O/Hypothalamus	Suspected role in the control of FSH secretion
Testosterone	Male: 300 – 1200 ng/dLFemale: 30 – 95 ng/dL	Steroid	T/Male accessory sex organs	Development of secondary sex characteristics, maturation, and normal function
Inhibin	Female (>16 yr):0 – 78 pg/mL	Polypeptide	T/Hypothalamus	Suspected role in the control of FSH secretion
Estrogens	Female estradiol (pmol/L): Early follicular: 100 – 200 Preovulatory: 500 – 1700 Luteal: 500 – 900 Postmenopausal: 70 – 200 Male: 0 – 283 pmol/LEstriol: Varies with gestational age (increases thru pregnancy)	Phenolic steroids	P/Female accessory sex organs	Development of secondary sex characteristics
Progesterone	Female: (nmol/L) Follicular: 2.0 – 4.5Luteal: 7.0 – 70.0	Steroid	P/Female accessory reproductive structure	Preparation of the uterus for ovum implantation, maintenance of pregnancy
Relaxin	-	Polypeptide	P/Uterus	Inhibition of myometrial contraction
Human chorionic gonadotropin (hCG) or choriogonadotropin	Female: (b -subunit)(Non-preg): <5 mIU/L	Glycoprotein‡ (a , 92 aa; ß, 144 aa)	P/Ovary AP/Testis	Ovulation, formation of corpora lutea, secretion of progesterone. Prolongation of corpus luteal function; suspected role in steroidogenesis during fetal life.Stimulation of interstitial tissue; secretion of androgens.
Human chorionic somatomammotropin (hCS) or human placental lactogen (hPL)	-	Protein (191 aa)	P/Mammary gland	Proliferation of mammary gland; initiation of milk secretion; antagonist of insulin action
Insulin, free	9 – 80 pmol/L	Polypeptide§	PAN/Most cells	Regulation of carbohydrate metabolism; lipogenesis
Glucagon	<190 ng/L	Polypeptide (29 aa)	PAN/Liver	Glycogenolysis
Pancreatic polypeptide (PP)	40 – 300 ng/L (fasting)	Polypeptide (36 aa)	PAN/Gastrointestinal tract	Increased gut motility and gastric emptying; inhibition of gallbladder contraction
Gastrin¶	Fasting: <90 ng/LRandom: <180 ng/L	Peptide (17 aa)	GI/Stomach	Secretion of gastric acid, gastric mucosal growth
Secretin	12 – 75 pg/mL	Polypeptide (27 aa)	GI/Pancreas	Secretion of pancreatic bicarbonate and digestive enzymes
Cholecystokinin-pancreozymin (CCK-PZ)¶	-	Polypeptide (33 aa)	GI/Gallbladder and pancreas	Stimulation of gallbladder contraction and secretion of pancreatic enzymes
Motilin	-	Polypeptide (22 aa)	GI/Gastrointestinal tract	Stimulation of gastrointestinal motility
Vasoactive intestinal peptide (VIP)¶	<20 pmol/L<50 pg/mL	Polypeptide (28 aa)	GI/Gastrointestinal tract	Neurotransmitter; relaxation of smooth muscles of gut and of circulation; increase of release of hormones and secretion of water and electrolytes from pancreas and gut
Gastric inhibitory polypeptide (GIP)	-	Polypeptide (42 aa)	GI/Gastrointestinal tract	Inhibition of gastric secretion and motility; increase of insulin secretion
Bombesin¶	-	Peptide (14 aa)	GI/Gastrointestinal tract	Stimulation of release of various hormones and pancreatic enzymes, smooth muscle contractions and hypothermia, changes in cardiovascular and renal function
Neurotensin¶	-	Peptide (13 aa)	GI/Gastrointestinal tract and hypothalamus (gut and brain)	Uncertain
Substance P (SP)¶	-	Peptide (11 aa)	GI/Gastrointestinal tract and brain	Sensory neurotransmitter, analgesic; increase in contraction of gastrointestinal smooth muscle; potent vasoactive hormone; promotion of salivation, increased release of histamine
1,25-(OH)2 Vitamin D	22.5 – 94.3 nmol/L	Sterol	K/Intestine K/Bone K/Kidney	Facilitation of calcium and phosphorus absorption Increase in bone resorption in conjunction with PTH.Increase in reabsorption of filtered calcium
Erythropoietin	12 – 28 U/L	Glycoprotein	K/Bone marrow	Stimulation of red cell formation
Insulin-like growth factor I	(ng/mL) 2m - 5y: 17 – 248 6 – 8 yr: 88 – 474 9-11 y Male: 110 – 565 9-11 y Fem: 117 – 771 12-15y Male: 202 – 957 12-15y Fem: 261 – 1096 16-24y M/F: 182 – 780 25-39y M/F: 114 – 492 40-54y M/F: 90 – 360³ 55 yr M/F: 71 – 290	Peptide (70 aa)	L/Most cells	Stimulation of cellular and linear growth
Insulin-like growth factor II	-	Peptide (67 aa)	L/Most cells	Insulin-like activity
Thymosin and thymopoietin	-	Peptides (49 and 28 aa)	THY/Lymphocytes	Maturation of T-lymphocytes
Atrial natriuretic factor (Atrial natriuretic peptide, ANF, ANP, Atriopeptin)	4 – 27 pmol/L	Peptide (28 aa)	HT/Vascular, renal, and adrenal tissue	Regulation of blood volume and blood pressure
Brain natriuretic factor (Brain natriuretic peptide, BNF, BNP)	5 – 99 pg/mL	Peptide (17 aa)	HT/Vascular, brain tissue	Regulation of blood volume and blood pressure
Parathyroid hormone-related peptide (PTH-RP)	<5 pmol/L	Peptide (141 aa)	MCT/Kidney, bone	Conjectural; PTH-like actions; tumor marker
Growth factors (e.g., epidermal growth factor, fibroblast growth factor, transforming growth factor family, platelet-derived growth factor, nerve growth factors)	-	Polypeptides	MCT/	Stimulation of cellular growth
Cytokines (e.g., interleukins 1 - 9, tumor necrosis factor, interferons)	-	Polypeptides	MLM/	Stimulation or inhibition of cellular growth

aa: Amino acid residues.
±Also produced by gastrointestinal tract.
‡Glycoprotein hormone composed of two dissimilar peptides. The a -chain is similar in structure or identical; the ß-chain differs for each hormone and confers specificity.
§Two chains linked by disulfide bonds: A, 21 aa; B, 30 aa.
¶Also produced in the brain.
>Source abbreviations: H = Hypothalamus, AP = Anterior Pituitary, PP = Posterior Pituitary, PG = Pineal Gland, TG = Thyroid Gland, PTG = Parathyroid Gland, AC = Adrenal Cortex, AM = Adrenal Mrdulla, O = Ovary, T = Testis, P = Placenta, PAN = Pancreas, GI = Gastrointestinal tract, K = Kidney, L = Liver, THY = Thymus, HT = Heart, MCT = Multiple Cell Types, MLM = Monocytes/Lymphocytes/Macrophages