Acid-Base Balance

An acid is any compound that can donate H+ when dissolved in water. A base is any compound that can donate OH- ions. A buffer system is a combination of a weak acid or base and its salt or conjugate that resists changes in pH. The human body has incredible mechanisms to maintain an acid-base balance. Changes in pH put the body in different physiological states that can cause an array of problems. Acidosis is when the pH falls below the reference range of 7.34. Alkalosis is when the pH increases above the reference range of 7.44.


The most important buffer system in the body is the bicarbonate (HCO3)/carbonic acid (H2CO3) system. Carbonic acid works to allow the human body to rid of toxic CO2 via respiration to maintain a normal pH of 7.4. There normally is a 20:1 ratio of bicarbonate to carbonic acid.

The red cells pick up CO2 from tissues and throughout its travel through the blood vessel its converted to carbonic acid. That carbonic acid is then broken down into bicarbonate and hydrogen. The excess hydrogen ions are buffered by hemoglobin. Bicarbonate leaves the red cell and goes into circulation. Bicarbonate enters the plasma through an exchange mechanism with chloride to maintain a state of electroneutrality in the cell. When the red cells reach the lung the hemoglobin will release the excess hydrogen ions by the binding of oxygen to hemoglobin. The excess hydrogen ions bind to bicarbonate to form carbonic acid. Carbonic acid then dissociates into H20 and CO2 which is expelled.

As mentioned above, an individual can be in a state of acidosis or alkalosis. This can be caused by ventilation and is called respiratory acidosis or respiratory alkalosis or it can either be caused by HCO3-. This is called metabolic acidosis or alkalosis.

Respiratory acidosis is an increase in PCO2. Conversely respiratory alkalosis is a decrease in PCO2. Metabolic acidosis is a loss of HCO3- or an addition of H+. Metabolic alkalosis is a loss of H+ or an increase of HCO3-. The body will naturally compensate for the pH changes. Some of the compensatory mechanisms are increasing respiration in metabolic acidosis. Hyperventilation increases the amount of CO2 that is expelled and raising the pH. In respiratory acidosis the kidney will increase its reabsorption of HCO3-.

Metabolic acidosis can be caused by multiple different disease states. Excessive loss of HCO3- by diarrhea can cause metabolic acidosis. Diabetic ketoacidosis can cause it. Other causes are ingestion of acids or renal tubular failure where there is no renal reabsorption of HCO3-.

Metabolic alkalosis is caused by excess or an overdose of HCO3-. Excessive vomiting causes a loss of hydrochloric acid with the stomach contents. Vomiting also results in hypokalemia and hyponatremia which are both positively charged ions (acids) leading to an increase in the pH. Excessive diuretic use can sometimes initially cause an increase in chloride, but most commonly results in hyponatremia and causing a contractile alkalosis.

Respiratory acidosis is most commonly caused by CO2 retention usually due to ventilation failure. Decreased cardiac output and hypotension also cause acidosis. Less blood is pumped to the heart so less CO2 is getting transported to the lungs to be expelled. Chronic lung conditions such as COPD result in an inability to ventilate properly and to expel CO2. Certain drugs cause depression of the respiratory center in the brain and can cause respiratory acidosis. Some of these drugs are barbiturates, opiates and ethanol (alcohol).

Respiratory alkalosis is primarily caused by hyperventilation (increased alveolar ventilation). This results in a decreased arterial PCO2. Any condition which decreases pulmonary compliance causes a sensation of dyspnea. Dyspnea is not a single sensation and there are at least three distinct sensations including air hunger, work/effort, and chest tightness. These sensations cause a state of hypoxia which is caused by the hyperventilation.



General Endocrinology

Hormones make up the endocrine system and act on almost every tissue in the body. Hormones are substances that are produced by a specialized cell that circulates in the blood. The best example of this is insulin which is secreted by the beta cells in the pancreas.


Credit for the photo goes to Pearson Education, Inc.

There are multiple forms of chemical signaling that hormones utilize. The first is autocrine where the cell targets itself. Signaling across gap junctions occurs when a specialized cell targets another cell that is connected via a gap junction. Paracrine is when the targeted cell is nearby. Endocrine which will be the primary focus for today is when the cell produces hormones or chemical signals that have to travel through the blood stream to act on distant cells. Depending on the receptor type to these hormones distinguishes the action it has on the recipient tissue or cell. Receptors can by cytoplasmic, ion channels, tyrosine kinase receptors, or a G-protein coupled receptor. There can also be different types of hormones. Protein hormones utilize calcium as a secondary messenger. The action potential of protein hormones is quick as opposed to steroid hormones. The action of steroid hormones is slow as steroids are not as membrane permeable as protein hormones. Its important to note that hormones are released in pulses. Each pulse has an amplitude and period.

The endocrine system needs feedback control loops to function properly. Negative control loops maintain hormonal balance. Positive control loops are actually what causes physiological changes in the tissues involved.

The endocrine system starts in the hypothalamus. The hypothalamus releases releasing hormones to stimulate the anterior and posterior pituitary to secrete effector hormones that act on various sites of the body.

The anterior pituitary otherwise known as the adenohypophysis secretes the majority of the hormones. Releasing hormones are secreted by the hypothalamic neurons into the hypothalamopituitary portal system. These hormones are then carried down the pituitary stalk by this portal system into the adenohypophysis. The anterior pituitary secretes adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), growth hormone (GH), prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). These all act on their respectable tissues/cells to secrete specific hormones. ACTH acts on the adrenal gland, which sits on top of the kidneys. The adrenal gland is responsible for secretion of catecholamines (epinephrine/norepinephrine) that influences the flight or fight response as well as glucocorticoids such as cortisol which have physiological effects throughout the entire body. TSH acts on the thyroid gland to secrete the thyroid hormones T3 and T4. These hormones also have wide-spread physiological function throughout the body. GH acts on the liver and influences bone, muscle, and tissue growth. PRL acts on the mammary glands such as the breast glands to stimulate growth and to start lactation. FSH and LH act on the testes of males to secrete inhibin and testosterone as well on the ovaries in females to secrete estrogen, progesterone, and inhibin. Decreased or elevated levels of any of these hormones can have detrimental effects on normal physiological processes. These discrepant levels can either be from primary disease (In the organ where the hormones are produced) or it can be secondary disease, i.e. from the hypothalamus, or pituitary.

Oxytocin and vasopressin (ADH) are the hormones secreted by the posterior or neurohypophysis pituitary. These are synthesized in the paraventricular supraoptic nuclei of the hypothalamus and are carried down the pituitary stalk by axonal transport. These hormones are then released into the general circulation in the neurohypophysis. Oxytocin works in females and males. It effects the uterine smooth muscle and mammary glands in females and in males it effects the smooth muscle in the ductus deferens and the prostate gland. Vasopressin or ADH promotes water retention in the distal tubules and collecting ducts of the kidneys. SIADH is excess ADH secretion and results in concentrated urine, and a low serum concentration. In other words there is low serum sodium which is bad! Diabetes insipidus on the other hand is deficiency in ADH.


Overview of the Immune System; Part One

The overall function of the immune system is to prevent or limit infection. It is essential for survival. Multiple organ systems, cells, and proteins are involved in the immune response. It is the most complex system that the human body has. The immune system is differentiated into two directions. Innate or non-specific immunity or Acquired (specific) immunity.

The Innate immune system consists of many components. The skin acts as a mechanical barrier and is typically the first line of defense against foreign substances. Mucous membranes consist of the bodies normal microbiota which compete with invading microbes. The mucous membranes are also lined with mucous and cilia which act in an elevator type motion to push foreign substances away. Physiological barriers such as temperature, pH and the complement system. The more acidic environment that a lower pH offers disrupts bacterial growth. Antimicrobial proteins and peptides are present in different epithelial locations in the body. Lysozymes are present in the tears and saliva and cleave the peptidoglycan cell wall present in bacteria. Secretory phospholipase A2 is present in the gut and can enter the bacterial cell and hydrolyze lipids in the cell membrane. Lectins target gram positive bacteria and forms pores in the membranes. Defensins integrate into the lipid and form pores which causes loss of membrane integrity. These defensins are present in PMNs (neutrophils) and lamellar bodies in the gut. Cathelicidins are present in neutrophils and macrophages in the lungs and intestines and distrupt membranes. Histatins are constitutively produced by the glands in the oral cavity and are active against pathogenic fungi.  Inflammation plays a huge role in the Innate immune system. Inflammation induces vasodilation and increase in capillary permeability causing an influx of immune cells like PMNs and macrophages. Inflammation can be observed by the four cardinal signs; rumor (redness), tumor (swelling), color (heat), and dolor (pain). The innate immune response is a rapid response.

Innate Immunity

The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b. There are three distinct pathways; the classical pathway, the lectin pathway, and the alternative pathway. All pathways generate a C3 convertase which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a. In the classical pathway the activated C1s cleaves C4 to C4a and C4b which binds to the microbial surface. C4b then binds C2, which is cleaved by C1s to C2a and C2b forming the C4b2b complex. C4b2b on the microbial surface is an active C3 convertase which cleaves C3 to C3a and C3b. This results in opsonization of the bacterial surface by C3b. The C4b2b3b complex is an active C5 convertase leading to the development of the membrane-attack complex. Each complement component (C4a/b, C2a/b, C3a/b) have different functions, but that is another discussion for another time. The lectin pathway of complement activation is when mannose-binding lectin (MBL) and ficolins recognize and bind to carbohydrates on the pathogen surface. Ficolins are similar to MBLs, but have a different carbohydrate binding domain. MBLs bind with high affinity to mannose and fucose residues. Conversely ficolins bind oligosaccharides containing acetylated sugars. When MBL binds to a pathogen surface MBL-associated serine protease (MASP)-2 is activated and cleaves C4 and C2 similar to the classical pathway. The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin (factor P) in the presence of pathogens. Properdin stabilizes the C3bBb complex. C3 undergoes spontaneous hydrolysis to C3(H20) which binds to factor B, allowing it to be cleaved by factor D into Ba and Bb. The C3(H20)Bb complex is essentially a C3 convertase which cleaves more C3 into C3a and C3b. C3b molecules result in opsonization of bacterial surfaces. Its important to recognize that all pathways lead to generation of a C5 convertase. C4b2a4b in the classical pathway, C4b2a3b in the lectin pathway, and C3b2Bb in the alternative pathway. C5 is cleaved into C5a/b that initiates the assembly of the terminal complement components. These are the terminal complement components that form the membrane-attack complex.


The membrane attack complex consists of an assembly of C6, C7, and C8. This complex undergoes a conformational change that results in polymerization of C9 which generates a large pore in the cell membrane. Host cells contain CD59 which prevents the assembly of the C9 molecules preventing the formation of the membrane-attack complex.

C3a, C4a, and C5a are unique in that these complement components are called anaphylatoxics. They initiate a local inflammatory response when systemic injection of these molecules occurs. They induce smooth muscle cell contraction and increased vascular permeability. They induce adhesion molecules and activate mast cells that invade and populate submucosal tissues to release inflammatory mediators such as histamine and TNF-a.

The Acquired or adaptive immune system is all about specificity. The Humoral branch of the acquired immune system is executed by the B lymphocytes that produce antibodies to specific antigens. The cell-mediated branch consists of antigen presenting cells (APC) such as the dendritic cells processing foreign substances and presenting proteins of those substances as antigens through the major histocompatibility complex (MHC) to CD8 T lymphocytes. These are cytotoxic T-cells that kill these foreign antigens. The acquired immune response is a slow response because it takes the body time to produce antibodies. An important aspect of the adaptive response is memory. Once antibodies have been produced to an antigen, these responses last and the time it takes to produce an antibody on subsequent exposures is rapidly decreased.

These two different systems work in conjunction to produce an adequate and sustained response. When foreign antigens are processed and expressed on the surface of APCs as MHC peptides, pro-inflammatory cytokines such as IL-12p70, IL-18, and IFN-a are secreted. These attract NK cells which primarily attack viruses as well as PMNs and macrophages that phagocytize these antigen peptides to destroy them. Adaptive immunity is also started with dendritic cells that also undergo antigen uptake and processing. This is also called the maturation signal. This signal is augmented by IFN-y and TNF-a secreted by macrophages and NK cells. These dendritic cells either present the antigen to B lymphocytes which are the antibody producers or they present the antigen to CD4/CD8 T-cell lymphocytes.

There are multiple classes of antibodies. IgD is typically expressed on B-cell lymphocytes during differentiation with IgM. IgD is also present in the serum in low concentrations. IgM is a pentamer and the largest immunoglobulin. It is the first antibody that is produced in the immune response. IgA is in high concentration in the mucosal linings, saliva, and tears. Typically part of first line defenses. IgG is present in high concentrations in the serum. IgG is unique in that it can cross the placenta. IgE is involved in allergic reactions. It binds to mast cells and basophils causing degranulation.


Non-Malignant Leukocyte Disorders

Non-Malignant simply means that it is localized to the leukocytes. Leukocytes are another name for the white blood cells, more specifically in the case of these disorders, the granulocytes. These disorders are fairly uncommon and are inherited. The following are ones that are found to distinct morphological features and affect the granulocyte functionality.

Alder Reilly Anomaly

Alder Reilly Anomaly is a recessive trait defect that causes incomplete degranulation of mucopolysaccharides. Large, darkly staining metachromatic cytoplasmic granules which can be seen and are partially digested mucopolysaccharides. These granules are characteristically referred to as Alder Reilly bodies. These can sometimes resemble toxic granulation, but it is important to note that in toxic granulation neutropenia, dohle bodies, and a left shift is seen. In Alder Reilly Anomaly none of those are present. Its also important to mention that the functionality of the granulocytes is not impaired.

Alder Reilly

Pelger Huet Anomaly

Pelger Huet Anomaly is an autosomal dominant syndrome characterized by decreased nuclear segmentation. This is caused by a mutation in the Lamin B receptor gene. Lamin B is an inner nuclear membrane protein that plays a role in normal leukocyte nuclear shape change during maturation. Morphological changes include hyposegmented neutrophils or neutrophil lobes connected by a thin nuclear filament. Pseudo or acquired PHA can be observed in the granulocytes in individuals with MDS, AML, or chronic myeloproliferative neoplasms.

Pelger Huet

Chediak Higashi Syndrome

Chediak Higashi Syndrome is characterized by an abnormal fusion of granules. These present as large and are dysfunctional. This is caused by a mutation in the LYST, or CHS1 gene that encodes for proteins involved in vesicle fusion or fission. The mutated protein causes loss of lysosomal movement and loss of phagocytosis. Thus leaving the individual susceptible to an increased number of infections without the innate immune system to fight them off. One of the characteristic findings is neutropenia.


May-Hegglin Anomaly

May-Hegglin Anomaly is a rare autosomal dominant platelet disorder that is characterized by variable thrombocytopenia, giant platelets, and dohle bodie like inclusions in the granulocytes. MHA is caused by a mutation in the MYH9 gene that causes a dysfunctional and disarray production of myosin heavy chains type IIa which affects the megakaryocytic maturation process as well as platelet fragmentation. Though most cases are clinically asymptomatic, the individual may present with mild bleeding tendencies.


Chronic Granulomatous Disease

In CGD, mutations in proteins that make up the NADPH oxidase complex. The mutations lead to failure of the phagocytes to generate the oxygen-dependent respiratory burst following phagocytosis. Normal phagocytosis of a microorganism leads to phosphorylation of cytosolic P47 and P67. Antibacterial neutrophil elastase and cathepsin G from the primary granules and cytochrome complex gp91 and gp22 from the secondary granules migrate to the phagolysosome. NADPH oxidase is formed when P47 and P67 combine with P40, RAC2, and the cytochrome complex. Majority of cases of CGD is due to mutations in P47 or gp91.

Leukocyte Adhesion Disorders

Normal recruitment of leukocytes to a site of inflammation involves capture of leukocytes from peripheral blood, followed by a process known as rolling along a vessel wall. Rolling involves binding of integrins to endothelial cell receptors which is high-affinity which ultimately leads to diapedesis of leukocytes into tissues from peripheral blood. With Leukocyte Adhesion disorders there are mutations that result in the inability of neutrophils and monocytes to adhere to endothelial cells, and the consequence is potentially fatal bacterial infections.

Leukocyte Adhesion Disorder I is caused by a mutation in the genes responsible for B2 integrin subunits. This leads to a decreased amount of the truncated form of the B2 integrin which is essential for endothelial cell adhesion. Patients typically present with neutrophilia, lymphadenopathy, splenomegaly, and characteristic skin lesions.

Leukocyte Adhesion Disorder II is caused by a mutation in the SLC35C1 gene. This gene encodes for a fucose transporter that moves fucose from the endoplasmic reticulum to the Golgi region. Fucose is needed for the synthesis of selectin ligands. The defective fucose transporter leads to the inability to produce functional selectins and causes defective leukocyte recruitment and reoccurring infections. LADII is much more rare than LADI. Clinical presentation is growth retardation, coarse facial features, and other physical deformities.

Leukocyte Adhesion Disorder III is even more rare than LADII and is caused by a mutation in the Kindlin-3 gene. The mutations impair leukocyte rolling and activation of B integrin. With LADIII there is also decreased platelet integrin GPIIbIIIa resulting in bleeding similar to that of Glanzmann Thrombasthenia.


Streptococcus Identification and Work Up

Streptococcus is a genus of coccus gram positive bacteria. They typically grow in chains of pairs as cell division occurs along a single axis in this family of bacteria. Most species in the streptococcus genus are oxidase negative, catalase negative, and most are facultative anaerobes. Many streptococcal species are not intact pathogenic and are part of the commensal human microbiota of the skin, mouth, intestine and upper respiratory tract. However, certain streptococcus species are the pathogenic agent for many cases of pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis.


Species of Streptococcus are classified based on their hemolytic properties. Alpha-hemolytic species cause oxidation of the iron within the red cells, giving a greenish color to appear on the blood agar. The bacteria produces hydrogen peroxide which oxidizes the hemoglobin to biliverdin. This is also referred to as incomplete hemolysis or partial hemolysis. Beta-hemolytic streptococci cause complete degradation of the red cells, appearing as wide clear areas surrounding the bacterial colonies. Gamma-hemolytic species cause no hemolysis.

The beta-hemolytic species are further classified by the Lancefield grouping. This is a serotype classification that describes the specific carbohydrate present on the bacterial cell wall. There are serotypes for Lancefield groups A-V. For times sake, the only ones that will be discussed are Group A, and Group B.

Alpha-Hemolytic Strep

Strep pneumoniae, often referred to as pneumococcus is the leading cause of bacterial pneumonia. It can also be the etiological agent for otitis media, sinusitis, meningitis and peritonitis. The viridian’s group of alpha-hemolytic streptococci are a large group of commensal organisms. They possess no Lancefield antigens (carbohydrates) and can or can not be hemolytic.


Beta-Hemolytic Strep

Streptolysin which is an exotoxin is the enzyme produced by the strep species that causes complete hemolysis of the red cells in the media. There are two types of streptolysin; Streptolysin O (SLO) and Streptolysin S (SLS). Streptolysin O is an oxygen-sensitive (labile) cytotoxin which is secreted by most of the Group A Streptococcus (GAS) which interacts with cholesterol in the membrane of the red cells. Streptolysin S is an oxygen-stable cytotoxin also produced by GAS species that affects the innate immune system of the host. It works in preventing the host immune system from clearing the infection.


Group A Strep

Group A strep, otherwise known as S. pyogenes is the causative agent for strep infections, invasive and non-invasive. The most common infection is pharyngitis, otherwise known as strep throat, impetigo, and scarlet fever. All these infections are non-invasive. Invasive GAS infections include necrotizing fasciitis, pneumonia, and bacteremia (bacteria present in the blood). Complications can arise from GAS infections. Rheumatic fever is a disease that affects the joints, kidneys, and the heart valves. It is the consequence of an untreated strep A infection. Antibodies created by the immune system cross-react with proteins in the body which causes an immune-mediated attack on the hosts own cells. Essentially an acquired auto-immune disease. GAS is also implicated in pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Autoimmune antibodies affect the basal ganglia causing rapid onset of psychiatric, motor, sleep and other neurological symptoms, primarily in the pediatric population.

GAS is diagnosed by either an rapid strep test or by culture.

Group B Strep

GBS, otherwise known as S. agalactiae, causes pneumonia and more importantly meningitis in neonates and the elderly. The American Congress of Obstetricians and Gynecologists now recommends that all women between 35 and 37 weeks gestation to be tested for group B strep. Those who test positive should be given prophylactic antibiotics during labor. The bacteria can cause premature rupture of the membranes during pregnancy and can colonize and cross the placenta to infect the fetus.

Laboratory diagnosis and Workup

After initial culture and preliminary identification of beta-hemolytic streptococcus is suspected there are further identification tests to make an accurate diagnosis. The first is by Lancefield antigen determination. Commercially available Lancefield antisera is used for the differentiation of beta-hemolytic species. These usually come as small kits and are directed to Lancefield groups A, B, C, F, and G. Antigen detection is demonstrated by agglutination by specific antibodies that are provided.

The PYR test is a rapid colormetric method most often used to distinguish S. pyogenes from other beta-hemolytic species. The PYR tests for the presence of the enzyme pyrrolidonyl aminopeptidase. This enzyme hydrolyzes L-pyrrolidonyl-b-naphthylamide (PYR) to B-naphthylamide, which produces a red color when a specific reagent is added. The test can be performed on paper strips that contain dried chromogenic substrates for the pyrrolidonyl aminopeptidase. S. pyogenes is PYR positive and in the case of an unknown organism displaying S. pyogenes morphology plus being PYR positive, it is acceptable to presumptively identify as S. pyogenes.

Bacitracin susceptibility is another test that can be used to differentiate S. pyogenes from other beta-hemolytic strep species. S. pyogenes has an increased susceptibility to bacitracin. A pure culture must be obtained and streaked on a sheep blood agar plate. A small disk containing 0.04 U of bacitracin is placed on the plate and incubated overnight at 35 degrees celsius in 5% CO2. A zone of inhibition surrounding the disk indicates susceptibility.

With the introduction and advancement of nucleic acid detection and serological methods in the laboratory it is now getting easier and faster to detect strep species without relying on culturing and further confirmation testing. It should be important to note that this does not replace the use of a culture or any of the further testing mentioned above. Serological testing relies on antibodies to anti-streptolysin O and anti-DNase B. The antibody levels against streptolysin O rise within one week of infection and peak around 3-6 weeks. DNase B is a nuclease among many that S. pyogenes uses to escape neutrophil extracellular nets. DNase B is specific for S. pyogenes. The antibody levels to DNase B rise post two weeks infection and reach maximum titers at around 6-8 weeks.

The Optochin test is used to differentiate alpha-hemolytic streptococci. This is either S. pneumoniae or strep viridians. S. pneumoniae species are sensitive to the chemical ethylhydrocupreine hydrochloride, otherwise known as optochin. Optochin disks, sometimes called P disks can be obtained from a commercial vendor. A pure culture is obtain and incubated to allow growth overnight. When growth has occurred, a subculture is plated and a P disk is placed on the agar and incubated overnight at 35 degrees celsius with 5% CO2. The zone of inhibition is then measured and a zone greater than 14 mm indicates sensitivity and allows for the presumptive diagnosis of pneumococci.

The bile solubility test is usually used as confirmatory test for S. pneumoniae that distinguishes it from other alpha-hemolytic species. Sodium deoxycholate (Bile) will lyse the cell wall of pneumococcal species.