25 March 2010

Chronic Pelvic Pain

Chronic Pelvic Pain
By:Jennifer Griffin, MD
M3 Student Clerkship Lecture
University of Nebraska Medical Center

Chronic Pelvic Pain
* Definition = Pain of apparent pelvic origin that has been present most of the time for 6 months
* Difficult to diagnose.
* Difficult to treat.
* Difficult to cure.
* =Physician and patient frustration.

Just because you’re a hammer doesn’t necessarily make every problem a nail.

Chronic Pelvic Pain
* Gynecologic
* Gastrointestinal
* Urologic
* Musculoskeletal/ Pelvic Floor
* Psychological
* United Kingdom data:
o Urinary dx 30.8%
o GI dx 37.7%
o Gynecologic 20.2%
o 25-50% have >1 dx
o MC Dx = endometriosis, adhesions, IBS, IC
Getting the History
* Nature of the Pain:
o Sharp, stabbing, colicky, burning?
o Where specifically is it located?
* Timing of the Pain:
o Does it come and go or is it constant?
o Does it occur with certain activities?
o Is it related to menses?
o Is it consistent and predictable?
* Modifying factors:
o Can you do anything to make it better/ worse?

Review of Systems
* Gynecologic:
o Association with menses?
o Association with sexual activity? (be specific)
o New sexual partners/ practices?
o Symptoms of vaginal dryness / atrophy?
o Other changes in menses?
o Use of contraceptives?
o Childbirth history and any associations?
o History of pelvic infections?
o History of other gyn problems/ surgeries?
* Gastrointestinal:
o Regularity of bowel movements?
o Diarrhea/ constipation/ flatus?
o Relief with defecation?
o History of hemorrhoids/ fissures/ polyps?
o Blood in stools, melena, or mucous?
o Nausea, vomiting, or appetite change?
o Weight loss?
* Urologic:
o Pain with urination?
o History of frequent / recurrent UTIs?
o Blood in urine?
o Symptoms of urgency or incontinence?
o Difficulty voiding?
* Musculoskeletal:
o History of trauma?
o Association with back pain?
o Other chronic pain problems?
o Association with position or activity?
* Psychological:
o History of abuse (verbal/ physical/ sexual)?
o Diagnosis of psychiatric disease?
o Association with life stressors?
o Exacerbated by life stressors?
o Family/ spousal support?
Chronic Pelvic Pain
* Diagnosis
o History and Physical
o Targeted imaging studies (U/S best for gyn evaluation)
o Laparoscopy
o Cystoscopy/ Colonoscopy
o Physical therapy evaluation
* Gynecologic Origin
o Endometriosis
o Primary Dysmenorrhea
o Leiomyomas
o Dyspareunia
o Vaginismus
o Adenomyosis
o Infectious causes
o Pelvic congestion syndrome
o Pelvic organ immobility
o Cancer
* ACOG Practice

Gyn Causes
* Cyclic:
o Primary dysmenorrhea
o Endometriosis
o Adenomyosis
o Mittleschmertz
* Non-cyclic:
o Pelvic masses
o Adhesions
o Infections
o Non-gyn causes
* Related to intercourse:
o Endometriosis
o Vaginismus
o Vaginal atrophy
o Musculoskeletal
o Any non-cyclic cause could be exacerbated.

Chronic Pelvic Pain: Cyclic
* Endometriosis
Chronic Pelvic Pain: Cyclic
* Endometriosis: Etiology
* Endometriosis: Classic Triad
* But may present with:
o Chronic pelvic pain
o Adnexal mass
* Endometriosis: Diagnosis
* Endometriosis:
* Endometriosis: Treatment
* Dysmenorrhea
* Leiomyomas
* Adenomyosis
* Dyspareunia
* Vaginismus

Chronic Pelvic Pain: Dyspareunia
* Pelvic Floor Muscle Spasm and Strain
Chronic Pelvic Pain: Non-cyclic
* Pelvic congestion syndrome
* Pelvic organ immobility
* Infectious causes
* Gynecologic malignancies
* Other Gynecologic origin:
* Treatment of Gynecologic Problems
* Urologic Origin, Level A:
o Bladder malignancy
o Interstitial Cystitis
o Radiation Cystitis
o Urethral Syndrome
* Bladder origin, Level B:
* Urologic origin, Level C:
* Urologic origin
* Urologic origin: Interstitial Cystitis
* Gastrointestinal Origin, Level A:
* Irritable Bowel Syndrome
* IBS Treatment
* Colon carcinoma
* Constipation
* Inflammatory Bowel Disease
* Gastrointestinal origin, Level C (no Level B):
* Musculoskeletal, Level A:
* Musculoskeletal origin, Level B:
* Musculoskeletal origin, Level C:
* Other Non-Gynecologic Origin, Level A:
* Psychological
* Other Non-Gynecologic origins, level B:
* Other Non-Gynecologic origin, Level C:
Clinical Pearl of Wisdom
Pelvic Pain Treatment Triad
* Medical treatment of most likely diagnosis.
* Psychiatric evaluation and treatment.
* Pelvic physical therapy.

Case Studies
Chronic Pelvic Pain
* Conclusions:
o Thorough history and physical
o Imaging and lab studies
o Many treatment options available

Chronic Pelvic Pain.ppt


Pelvic Pain – Dysmenorrhea and Endometriosis

Pelvic Pain – Dysmenorrhea and Endometriosis

* A 20 y.o. woman presents to her gynecologist with a 4 year history of increasing lower abdominal pain with her menses. The pain begins on the first day of her menses and lasts 2-3 days. She also complains of lower back pain and nausea. Menarche occurred at the age of 13 and her menses occur every 28 days and last 5 days. Physical and pelvic exam are normal.

* How is dysmenorrhea diagnosed? How is it distinguished from other types of pelvic pain?
* What is the pathophysiology of dysmenorrhea?
* What are reasonable approaches to treatment?

* Dysmenorrhea – severe, painful cramping sensation in the lower abdomen often accompanied by other symptoms – sweating, tachycardia, headaches, n/v, diarrhea, tremulousness, all occurring just before or during menses
- Primary: no obvious pathologic condition, onset < 20 years old - Secondary: associated with pelvic conditions or pathology Primary Dysmenorrhea * Pathogenesis: elevated PG F2α in secretory endometrium (increased uterine contractility) * Treatment: NSAIDs – PG synthetase inhibitors – 1st line treatment of choice * Other treatment options: OCPs, other analgesics Secondary Dysmenorrhea * Etiologies - Cervical Stenosis - Endometriosis and Adenomyosis - Pelvic Infection - Adhesions - Pelvic Congestion - Stress and Tension * Cervical Stenosis - Severe narrowing of cervical canal may impede menstrual outflow – congenital or iatrogenic - can cause an increase in intrauterine pressure during menses - can lead to endometriosis * Cervical Stenosis - Hx – scant menstrual flow, severe cramping throughout menses - Dx – inability to pass a thin probe through the internal os OR HSG demonstrates thin cx canal - Tx – cervical dilation via D&C or laminaria placement * Pelvic Congestion - Due to engorgement of pelvic vasculature - Hx – burning or throbbing pain, worse at night and after standing - Dx – Laparoscopic visualization of engorgement/varicosities of broad ligament and pelvic sidewall veins Evaluation of Pelvic Pain * Detailed history, targeted physical exam, labs (UA, UCx, CBC, HCG, tumor markers), diagnostic imaging studies (US, MRI, CT) as appropriate * Consider age of patient * “OLDCAAR”: onset, location, duration, context, associated sx, aggravating/relieving factors * Temporal characteristics: cyclic (e.g. dysmenorrhea), intermittent (e.g. dyspareunia), non-cyclic * Risk factors * GYN and Non-GYN causes DDx Pelvic Pain - GYN * GYN - Uterus - fibroids, adenomyosis, endometritis - Fallopian tubes - PID/salpingitis, hydrosalpinx, ectopic - Ovaries - cysts – functional, pathological, TOA, torsion; mittleschmerz - Other - endometriosis, adhesions, IUD/infection, severe prolapse DDx Pelvic Pain – Non-GYN * Urologic - UTI/urethritis, interstitial cystitis (IC), OAB, urethral diverticulum, nephrolithiasis, malignancy * GI - constipation, IBS, IBD (Crohn’s, UC), bowel obstruction, diverticulitis, malignancy, appendicitis * Musculoskeletal - trigger points, fibromyalgia, hernias, neuralgia, low back pain * Other - psychiatric – depression, somatization; abdominal cutaneous nerve entrapment in surgical scar; celiac disease Case 1 * At the age of 30, the patient presents with a 2 year history of infertility. Her menses are still regular but she has 2-3 days of spotting before her menses are due. She also complains of pain with intercourse and pelvic pain. In reviewing the patient’s history, the gynecologist notes that over the past year the patient was repeatedly treated by her internist with antibiotics for recurrent microscopic hematuria. * What is the most likely diagnosis? * What are the main theories regarding the pathogenesis in this case? * How would you evaluate and treat this patient? Endometriosis - Symptoms * Variable and unpredictable - asymptomatic - dysmenorrhea - CPP - deep dyspareunia - sacral backache w/ menses - dysuria +/- hematuria (bladder involvement) - dyschezia/hematochezia (bowel involvement) Endometriosis – Physical Exam * Uterosacral nodularity * Adnexal mass (endometrioma) * Normal exam Endometriosis - Incidence * 7-10% of general population * 20-50% of infertile women * 70-85% in women w/ CPP * No racial predisposition * +Familial association with almost 10x increased risk of endometriosis if affected 1st degree relative Endometriosis - Pathogenesis * Retrograde menstruation (Sampson) * Hematogenous or lymphatic spread (Halban) * Coelomic metaplasia (Meyer/Novack) * Iatrogenic dissemination * Immunologic defects (Dmowski) * Genetic predisposition Endometriosis - Pathogenesis * Retrograde menstruation (Sampson’s theory) - Monkey experiments – sutured cervix closed to create outflow obstruction caused development of endometriosis - Clinical observation of retrograde menstrual flow during laparoscopy in humans - Increased risk of endometriosis in women with cervical/vaginal atresia, other outflow obstruction - Increased risk with early menarche, longer and heavier flow - Decreased risk with decreased estrogen levels e.g. exercise-induced menstrual disorders, decreased body fat, + tobacco use Endometriosis - Pathogenesis * Hematogenous or lymphatic spread - Endometriosis found in remote sites – lung, nose, spinal cord, pelvic lymph nodes. Endometriosis - Pathogenesis * Coelomic metaplasia - Mullerian ducts are derived from coelomic epithelium during fetal development - Hypothesize that coelomic epithelium retains ability for multipotential development - Endometriosis seen in prepubertal girls, women w/ congenital absence of the uterus, and RARELY in men Endometriosis - Pathogenesis * Iatrogenic dissemination - Endometriosis has been found in cesarean section scar * Immunologic defects * Genetic predisposition - polygenic, multi-factorial Endometriosis - Diagnosis * Laparoscopy with biopsy proven histologic diagnosis – standard for dx of endometriosis * Empiric medical treatment with improvement in symptoms * CA 125 – NOT considered to be of clinical utility * Imaging – US, MRI, CT – only useful in the presence of pelvic or adnexal masses (endometriomas) * Laparoscopy with biopsy proven histologic diagnosis – standard for dx of endometriosis - Extent of visible lesions do not correlate with severity of sx, but depth of infiltration of lesions seems to correlate best with pain severity - classic powder-burn lesions, endometriomas - lesions can be red, clear or white – more commonly seen in adolescents * Endometrial epithelium * Endometrial glands * Endometrial stroma * Hemosiderin-laden macrophages 2 or more of the following histologic features are criteria for Dx: * Imaging – US, MRI, CT – only useful in the presence of pelvic or adnexal masses (endometriomas) - on US, endometriomas appear as cysts that contain low-level homogeneous internal echoes consistent with old blood (ddx includes hemorrhagic cysts) Endometriosis - Treatment * Medications - Progestins - OCPs – continuous vs. cyclic – if no relief in 3 months, consider tx with Depo Provera or GnRH agonist - NSAIDs - GnRH agonists – most expensive - Danazol – appears to be as effective as GnRH agonist for pain relief but with increased side-effects * GnRH agonists – create a state of relative estrogen deficiency – vasomotor side effects and potential decrease in bone density - 12-month course of GnRH agonist therapy associated with 6% decrease in bone density - No data regarding extended treatment with GnRH agonists beyond 1 year * Add-back therapy is advocated for women undergoing long-term therapy (i.e. > 6 months)
* Some evidence to suggest that immediate add-back therapy may result in even less bone loss
- Add-back regimens: progestins alone, progestins + bisphosphonates, low-dose progestins + estrogens, pulsatile PTH

Endometriosis – Treatment Considerations in Adolescents
* GnRH treatment is NOT recommended for patients < 18 years because the effects of these medications on bone formation and long-term bone density have not been adequately studied * Depo provera used for longer than 2 years has been shown to decrease bone density in adolescents – FDA warning against long-term use * If no improvement in symptoms after 3 months of empiric treatment with NSAIDs and OCPs, diagnostic laparoscopy should be offered Endometriosis - Treatment * Surgery - Laparoscopic laser vaporization vs. cauterization vs. excision - Ovarian cystectomy for endometrioma - Hysterectomy +/- BSO * Medications vs. Surgery - Lack of data to support surgery vs. medical treatment for tx of pain symptoms due to endometriosis - Starting with empiric medical therapy is appropriate - Offer GnRH agonist therapy if initial medical treatment with OCPs and NSAIDs not helping - Cost of comparing empiric medical management with definitive surgical diagnosis is difficult to assess, but 3 months of empiric treatment is less than a laparoscopic procedure .... Pelvic Pain – Dysmenorrhea and Endometriosis.ppt


Urinary Tract Infections

Urinary Tract Infections
By:Lourdes Lozano Vargas

Urinary Tract Infections
* Leading cause of morbidity and health care expenditures in persons of all ages.
* An estimated 50 % of women report having had a UTI at some point in their lives.
* 8.3 million office visits and more than 1 million hospitalizations, for an overall annual cost > $1 billion.

Acute Uncomplicated Cystitis
* Sexually active young women.
* Causes: anatomy and certain behavioral factors, including delays in micturition, sexual activity, and the use of diaphragms and spermicides tract.
* Aggressive diagnostic work-ups are unwarranted in young women presenting with an uncomplicated episode of cystitis.

Acute Uncomplicated Cystitis
* The microbiology is limited to a few pathogens.
* 70%- 85% are caused by Escherichia coli
* 5-20%are caused by coagulase-negative Staphylococcus saprophyticus
* 5-12% are caused by other Enterobacteriaceae such as Klebsiella and Proteus.

Acute Uncomplicated Cystitis
* Clinical Features: dysuria, frequency, urgency, suprapubic pain, hematuria.
o Fever >38C, flank pain, costovertebral angle tenderness, and nausea or vomiting suggest upper tract infection.

Acute Uncomplicated Cystitis
* Diagnosis: direct history and PE
* PE: Temperature, abdominal exam, assessment of CVA tenderness, pelvic exam.
o H/o STD’s, new sexual partner, partner with urethral symptoms, gradual onset.

Acute Uncomplicated Cystitis
* Guidelines for tx of acute cystitis recommend empiric antibiotic tx.
* Unnecessary antibiotic use??
* Clinical criteria for Dx:

Dysuria, presence of > trace urine leukocytes, and presence of nitrites or...
Dysuria and frequency in the absence of vaginal discharge.
Acute Uncomplicated Cystitis
* UA: Evaluation of midstream urine for pyuria.
o White blood cell casts in the urine are Dx of upper tract infection.
* Urine Culture: Not necessary
o Warranted in: Suspected complicated infection, persistent symptoms following tx, symptoms recur < 1 mo after tx. Acute Uncomplicated Cystitis * Urine dipsticks: o Leukocyte esterase (pyuria), sensitivity 75-90%, specificity 95% o Nitrite (Enterobacteriacea), sensitivity 35-85%, specificity 95%, false positive with phenazopyridine, beets. o Microscopic evaluation for pyuria or a culture is indicated in pt with negative leukocyte esterase that have urinary symptoms. Acute Uncomplicated Cystitis * Susceptibility: o E.coli o S.saprophyticus Acute Uncomplicated Cystitis * Treatment: o Short course vs. prolonged tx + Short course preferred except with beta-lactam agents o TMP-SMX (160/800mg BID x 3) first-line tx if: no allergy to the drug, no antibiotics in the past 3 mo, no recent hospitalization. o Nitrofurantoin (100mg BID x 5 days) o Analgesia: Phenazopyridine 200mg TIDx2 Acute Urethral Syndrome * Acute symptomatic women with dysuria and frequency with a midstream culture containing < 10(5) CFU/mL. * > 10(2) CFU/mL in women with acute symptomatic pyuria = UTI
* Tx as an uncomplicated UTI
* Mycoplasma genitalium, Ureaplasma urealyticum

Acute Complicated Cystitis
* UTI when/with structural, functional or metabolic abnormalities (polycystic, solitary, transplant kidney;DM, CRF, indwelling cath, neurogenic bladder) or elderly, male, child, pregnant or h/o recurrent UTI)
* E.coli accounts for fewer than one third of complicated cases.
* Clinically, the spectrum of complicated UTIs may range from cystitis to urosepsis with septic shock.

Acute Complicated Cystitis
* Urine culture and susceptibility are necessary.
* These infections are usually associated with high-count bacteriuria (> 10(5) CFU/mL).
* MO: Proteus, Klebsiella, Pseudomonas, Serratia, and Providencia, enterococci, staphylococci and fungi AND E.coli

Acute Complicated Cystitis
* Empiric therapy for these patients should include an agent with a broad spectrum of activity against the expected uropathogens: fluoroquinolone, ceftazidime, cefepime, aztreonam, imipenem-cilastatin. (Obtain Ucx prior to Tx)
* Tx x 7-14 days
* Follow-up urine culture should be performed within 14 days after treatment???

Recurrent Cystitis
* Up to 27% of young women with acute cystitis develop recurrent UTIs.
* The causative organism should be identified by urine culture.
* Relapse: infection with the same organism (multiple relapses = complicated UTIs).
* Recurrence: infection with different organisms.

Recurrent Cystitis
* >3 UTI recurrences documented by urine Cx within one year can be managed using one of three preventive strategies:
* Acute self-treatment with a three-day course of standard therapy.
* Postcoital prophylaxis with one-half of a TMP-SMX double-strength tablet (80/400 mg).
* Continuous daily prophylaxis TMP-SMX one-half tablet per day (40/200 mg); nitrofurantoin 50 to 100 mg per day; norfloxacin 200 mg per day.

Uncomplicated Pyelonephritis
* Suspect if:
o Cystitis-like illness and accompanying flank pain
o Severe illness with fever, chills, nausea, vomiting, abdominal pain
o Gram-negative bacteremia.

Uncomplicated Pyelonephritis
* DX: Clinical, confirm with:
o UA: pyuria and/or WBC casts
o UCx with > 10 (5) CFU/mL (80%)
* Tx: 14 days total
o Oral: TMP/SMX, fluoroquinolones
o IV: 3rd gen cephalosporin, aztreonam, quinolones, aminoglycoside

Uncomplicated Pyelonephritis
* Pt with symptoms after 3 days of appropriate antimicrobial tx should be evaluated by renal US or CT for obstruction or abscess.

UTI in Men

* At risk: Older men with prostatic disease, UT instrumentation, anal sex, or partner colonized with uropathogens.
* UCx: 10 (3) CFU/mL sensitivity and specificity 97%.
* Additional studies?
o Not necessary in young healthy men who have a single episode.

UTI in Men
* Tx:
o Uncomplicated cystitis:
+ TMP/SMX or fluoroquinolones x 7 days
o Complicated cystitis:
+ Fluoroquinolones x 7-14 days
o Bacterial prostatitis:
+ Fluoroquinolone x 6-12 weeks

Catheter-Associated UTI
* Risk of bacteriuria is ~ 5%/day (long term catheter bacteriuria is inevitable).
* 40% of nosocomial infections
* Most common source of gram-negative bacteremia.
* Dx: Ucx 10 (2) CFU/mL
o MO: E.coli, Proteus, Enterococcus, Pseudomona, Enterobacter, Serratia, Candida

Catheter-Associated UTI
* Mild to mod: oral quinolones10-14days
* Severe infection: IV/oral 14-21days
* Asymptomatic bacteriuria in pt with an indwelling Foley should not be Tx unless they are immunosuppressed, have risk of bacterial endocarditis or pt who are about to undergo urinary tract instrumentation.

Asymptomatic Bacteriuria
* UCx: > 10(5)CFU/mL with no symptoms
* Three groups of pt with asymptomatic bacteruria have been shown to benefit from tx:
o Pregnant
o Renal transplant
o Pt who are about to undergo urinary tract procedures.

Pregnant patients

* Asymptomatic bacteriuria: two consecutive voided urine specimens with isolation of the same bacterial strain >10(5) or a single cath urine specimen.
o Nitrofurantoin 100mg BID x 5-7 days
o Amoxi/Clav 500mg BID or 250 TID x 7days
o Fosfomycin 3g PO x 1

Interstitial Cystitis
* Frequency, urgency, urge incontinence with periurethral and suprapubic pain on bladder filling that is improved by voiding. Terminal hematuria may be present.
* Etiology. Unclear (autoimmune, altered glycosaminoglycal layer, allergic)

Interstitial Cystitis
* TX
o Refer to urology for cystoscopy.
o Dietary modifications
o Behavioral modifications
o Rx:
+ Pyridium
+ Pentosan polysulfate 100mg TID x 6mo to 2 years.
+ Amitriptyline 10-75mg QHS

Interstitial Cystitis
* Intravesical therapies
o Dimethyl Sulfoxide instillations q1-2 wks
o BCG instilled q1wk x 6-8 wks
o Hyaluronic acid instilled q1wk x 4-6wk.


Urinary Tract Infections.ppt


Oral Cavity

Oral Cavity
By:Robert Scranton© 2008

The Tissues
Lining Mucosa
Masticatory Mucosa
* NKSS (nonkeratinized stratified squamous)
* Lamina Propria- loose CT w/ collagen bundles
o Mucous and serous glands
o Fordyce Spots
* Location?
* KSS/PKSS (keratinized/parakeratinized stratified squamous)
* Variable Lamina Propria
* Location?

Lining Mucosa
Don’t forget the soft palate
Diagrams are important

* vermillion zone
* Hair follicle
* Epithelium, what type?
* Skeletal muscle
o what is the name?

Special Mucosa
* Filiform
o Most abundant
o Dorsal surface
* Fungiform
o Occasional tasebuds, CN-VII
* Vallate
o 8-12 along sulcus terminalis
o Crypt
o Serous glands of Von Ebner
o CN- IX, taste buds
* Foliate
o Dorsolateral surfaces, taste buds?

Identify filiform and fungiform
Vallate/ circumvallate

* We origionally have __ baby (________) teeth. Adults have ___ teeth.
* What are the three cell types that form the teeth and what parts do they form?
* What do dentin and enamel have in common?
* Which is acellular?

Mesenchymal CT pulp cavity
Odontoblasts (mesenchyme) Dentin
ameloblasts (ectoderm) Enamel
Ca2+ Hydroxyapatite (calcified organic Matrix
* The little tubules in the teeth, what is their story?
* Damage to What three things can lead to loss of a tooth?
* Dentinal tubules- the tubule that the cytoplasmic process of odontoblasts extend through for nociception
* Canaliculi- the tubules that cementocytes use to maintain cementum
* Bony Socket
* Peridontal ligament
* Cementum

* Alveolar Bone
* Free Gingiva
* Attached Gingiva
* Alveolar Mucosa
* Gingival Ligament
* Gingival Sulcus
* Alveolar Bone
* Dentin
* Peridontal ligament
* Pulp Cavity
* Gingiva
* Odontoblasts
* Predentin
* Dentin
* Cementocytes
* Peridontal Ligament

Salivary Glands
* AKA minor
* Serous
* Mucous
* Mixed
* Means w/in lamina propria
* AKA Major
* Serous
* Serousmucous
* Outside oral cavity
* Has large ducts

Important words
* Serous amylase
* Serous demilunes bacteriolytic lysozyme
* IgA bacteriostatic, resistant to degradation
* Nasopharynx Respiratory Epithelium
* Oropharynx lining mucosa, NKSS
* Laryngopharynx transitional zone so KSS, NKSS

Oral Cavity.ppt


Chemical composition and functions of saliva

Chemical composition and functions of saliva
By:Dennis E. Lopatin, Ph.D.

Chronology of defining salivary components and functions
* Beginning in 1950’s whole saliva evaluated (antimicrobial properties, role in microbial attachment, mineralization, taste, lubrication)
* Secretions of major glands (parotid and submandibular/sublingual)
* In 1970’s individual components isolated and biochemically characterized
* In mid-1980’s beginning to map functional domains (peptide synthesis and recombinant approaches)

Major salivary components
Mucin 1 (MG1)
Mucin 2 (MG2)
Carbonic anhydrases
Proline-rich proteins

Current concepts regarding the functional features of salivary macromolecules
* Recent structure/function studies have identified general principles regarding function
* Based on in vitro studies of purified molecules
* Additional studies required to evaluate concepts in situ

Conformational requirements
* Conformation or shape of a molecule is critical for its biological function
* Examples
o Proline-rich proteins interact with A. viscosus and St. gordonii only when adsorbed onto mineralized surface
o Statherins and histatins require -helical conformation
o Human salivary amylase require 5 inter-chain disulfide bonds

Lubrication &Viscoelasticity
Carbonic anhydrases,
Mucins, Lipase
Histatins, Proline rich proteins,
Mucins, Statherins
Cystatins, Mucins,
Proline-rich proteins, Statherins
Amylases, Cystatins,
Histatins, Mucins,
* Saliva has built-in redundancy in regard to its protective functions.
* Example - Many salivary molecules can inhibit the precipitation of calcium phosphate salts.
o strong inhibitors such as statherin and acidic proline-rich proteins
o moderate inhibitors such as histatins and cystatins
o weak inhibitors such as mucins and amylase

* A molecule may have both protective and detrimental properties - “double-edged sword”.
* May depend on molecule’s location or site of action
o Amylases
+ In solution, they facilitate clearance of viridans streptococci
+ Adsorbed to tooth surface, they can promote adherence of these bacteria and digest starch to dietary maltose and production of acid
o Statherin and acidic proline-rich proteins
+ At enamel surface, they play an important role in mineralization by inhibiting the formation of primary and secondary calcium phosphate salts. When adsorbed to the enamel surface, they promote attachment of cariogenic microorganisms.

* Functional relationships exist between different molecules in saliva
* Two types of complexing (covalent and non-covalent)
o homotypic (between similar molecules)
o heterotypic (between different molecules)
* Example: Mucins
o homotypic complexes necessary for lubrication and viscoelastic properties
o heterotypic complexes with sIgA, lysozyme and cystatins concentrate these anti-microbials at tissue interfaces
Salivary Protein Functions
* Lack precise folded structure of globular proteins
* Asymmetrical molecules with open, randomly organized structure
* Polypeptide backbone (apomucin) with CHO side-chains
* Side-chains may end in negatively charged groups, such as sialic acid and bound sulfate
* Hydrophillic, entraining water (resists dehydration)
* Unique rheological properties (e.g., high elasticity, adhesiveness, and low solubility)
* Two major mucins (MG1 and MG2)

Mucin Functions
* Tissue Coating
o Protective coating about hard and soft tissues
o Primary role in formation of acquired pellicle
o Concentrates anti-microbial molecules at mucosal interface
* Lubrication
o Align themselves with direction of flow (characteristic of asymmetric molecules)
o Increases lubricating qualities (film strength)
o Film strength determines how effectively opposed moving surfaces are kept apart
* Aggregation of bacterial cells
o Bacterial adhere to mucins may result in surface attachment, or
o Mucin-coated bacteria may be unable to attach to surface
* Bacterial adhesion
o Mucin oligosaccharides mimic those on mucosal cell surface
o React with bacterial adhesins, thereby blocking them

* Calcium metalloenzyme
* Hydrolyzes (1-4) bonds of starches such as amylose and amylopectin
* Several salivary isoenzymes
* Maltose is the major end-product (20% is glucose)
* “Appears” to have digestive function
* Why is it also present in tears, serum, bronchial, and male and female urogenital secretions?
* A role in modulating bacterial adherence?

Lingual Lipase
* Secreted by von Ebner’s glands of tongue
* Involved in first phase of fat digestion
* Hydrolyzes medium- to long-chain triglycerides
* Important in digestion of milk fat in new-born
* Unlike other mammalian lipases, it is highly hydrophobic and readily enters fat globules

* Calcium phosphate salts of dental enamel are soluble under typical conditions of pH and ionic strength
* Supersaturation of calcium phosphates maintain enamel integrity
* Statherins prevent precipitation or crystallization of supersaturated calcium phosphate in ductal saliva and oral fluid
* Produced by acinar cells in salivary glands
* Also an effective lubricant

Proline-rich Proteins (PRPs)
* Like statherin, PRPs are also highly asymmetrical
* Inhibitors of calcium phosphate crystal growth
* Inhibition due to first 30 residues of negatively-charged amino-terminal end
* Present in the initially formed enamel pellicle and in “mature” pellicles

Role of PRPs in enamel pellicle formation
* Acquired enamel pellicle is 0.1-1.0 µm thick layer of macromolecular material on the dental mineral surface
* Pellicle is formed by selective adsorption of hydroxyapatite-reactive salivary proteins, serum proteins and microbial products such as glucans and glucosyl-transferase
* Pellicle acts as a diffusion barrier, slowing both attacks by bacterial acids and loss of dissolved calcium and phosphate ions

Remineralization of enamel and calcium phosphate inhibitors
* Early caries are repaired despite presence of mineralization inhibitors in saliva
* Sound surface layer of early carious lesion forms impermeable barrier to diffusion of high mol.wt. inhibitors.
* Still permeable to calcium and phosphate ions
* Inhibitors may encourage mineralization by preventing crystal growth on the surface of lesion by keeping pores open

Calculus formation and calcium phosphate inhibitors
* Calculus forms in plaque despite inhibitory action of statherin and PRPs in saliva
* May be due to failure to diffuse into calcifying plaque
* Proteolytic enzymes of oral bacteria or lysed leukocytes may destroy inhibitory proteins
* Plaque bacteria may produce their own inhibitors

Calcium phosphate precipitation inhibitors and plaque
* Statherin and PRPs might be expected to occur in plaque, have not been detected
* Plaque bacteria produce calcium phosphate inhibitors
* Might be necessary to prevent calcification of bacteria -- happens with dead cells
* Immobilized crystal growth inhibitors can function as nucleators of crystal growth
* Immobilization may occur in plaque, encouraging calculus formation

Interaction of oral bacteria with PRPs and other pellicle proteins
* Several salivary proteins appear to be involved in preventing or promoting bacterial adhesion to oral soft and hard tissues
* PRPs are strong promoters of bacterial adhesion
o Amino terminal: control calcium phosphate chemistry
o Carboxy terminal: interaction with oral bacteria
* Interactions are highly specific
o Depends on proline-glutamine carboxy-terminal dipeptide
o PRPs in solution do not inhibit adhesion of bacteria

These anti-microbial proteins will be discussed in a later lecture
* Secretory Immunoglobulins
* Lactoferrin
* Lysozyme
* Sialoperoxidase
* Cystatins
* Histatins

Summary - Clinical Highlights
* Understanding of salivary mechanisms at fundamental level a prerequisite for
o effective treatment of salivary gland dysfunctions
o modulation of bacterial colonization
o development of artificial saliva other “cutting edge” approaches to salivary dysfunctions and diseases

Chemical composition and functions of saliva.ppt


Salivary Glands

Salivary Glands

Major glands
* Parotid: so-called watery serous saliva rich in amylase, proline-rich proteins
o Stenson’s duct
* Submandibular gland: more mucinous
o Wharton’s duct
* Sublingual: viscous saliva
o ducts of Rivinus; duct of Bartholin

Minor glands
* Minor salivary glands are not found within gingiva and anterior part of the hard palate
* Serous minor glands=von Ebner below the sulci of the circumvallate and folliate papillae of the tongue
* Glands of Blandin-Nuhn: ventral tongue
* Palatine, glossopalatine glands are pure mucus
* Weber glands

* Protection
o lubricant (glycoprotein)
o barrier against noxious stimuli; microbial toxins and minor traumas
o washing non-adherent and acellular debris
o formation of salivary pellicle
+ calcium-binding proteins: tooth protection; plaque
* Buffering (phosphate ions and bicarbonate)
o bacteria require specific pH conditions
o plaque microorganisms produce acids from sugars


* Digestion
o neutralizes esophageal contents
o dilutes gastric chyme
o forms food bolus
o brakes starch
* Antimicrobial
o lysozyme hydrolyzes cell walls of some bacteria
o lactoferrin binds free iron and deprives bacteria of this essential element
o IgA agglutinates microorganisms
* Maintenance of tooth integrity
o calcium and phosphate ions
+ ionic exchange with tooth surface
* Tissue repair
o bleeding time of oral tissues shorter than other tissues
o resulting clot less solid than normal
o remineralization
* Taste
o solubilizing of food substances that can be sensed by receptors
o trophic effect on receptors

Embryonic development
* The parotid: ectoderm (4-6 weeks of embryonic life)
* The sublingual-submandibular glands: endoderm
* The submandibular gland around the 6th week
* The sublingual and the minor glands develop around the 8-12 week
* Differentiation of the ectomesenchyme
* Development of fibrous capsule
* Formation of septa that divide the gland into lobes and lobules

Serous cells
* Seromucus cells=secrete also polysaccharides
* They have all the features of a cell specialized for the synthesis, storage, and secretion of protein
o Rough endoplasmic reticulum (ribosomal sites-->cisternae)
o Prominent Golgi-->carbohydrate moieties are added
Secretory granules-->exocytosis
* The secretory process is continuous but cyclic
* There are complex foldings of cytoplasmic membrane
* The junctional complex consists of:
o Tight junctions (zonula occludens)-->fusion of outer cell layer
o Intermediate junction (zonula adherens)-->intercellular communication
o Desmosomes-->firm adhesion

Mucous cells
* Production, storage, and secretion of proteinaceous material; smaller enzymatic component
-more carbohydrates-->mucins=more prominent Golgi
-less prominent (conspicuous) rough endoplasmic reticulum, mitochondria
-less interdigitations

Formation and Secretion of Saliva
* Primary saliva
o Serous and mucous cells
o Intercalated ducts
* Modified saliva
o Striated and terminal ducts
o End product is hypotonic

Macromolecular component
* Synthesis of proteins
* RER, Golgi apparatus
* Ribosomes RER posttranslational modification (N- & O-linked glycosylation) Golgi apparatus Secretory granules
* Exocytosis
* Endocytosis of the granule membrane

Fluid and Electrolytes
* Parasympathetic innervation
* Binding of acetylcholine to muscarinic receptors
o Activation of phospholipase IP3 release of Ca2+ opening of channels K+, Cl- Na+ in
o K+ and Cl- in
o Also another electrolyte transport mechanism through HCO3-
* Noepinephrine via alpha-adrenergic receptors
o Substance P activates the Ca2+

Myoepithelial cells
* One, two or even three myoepithelial cells in each salivary and piece body
* Four to eight processes
* Desmosomes between myoepithelial cells and secretory cells
* Myofilaments frequently aggregated to form dark bodies along the course of the process

Myoepithelial cells
* The myoepithelial cells of the intercalated ducts are more spindled-shaped and fewer processes
* Ultrastructurally very similar to that of smooth muscle cells
* Functions of myoepithelial cells
o Support secretory cells
o Contract and widen the diameter of the intercalated ducts
o Contraction may aid in the rupture of acinar cells of epithelial origin

Intercalated Ducts
* Small diameter
* Lined by small cuboidal cells
* Nucleus located in the center
* Well-developed RER, Golgi apparatus, occasionally secretory granules, few microvilli
* Myoepithelial cells are also present
* Intercalated ducts are prominent in salivary glands having a watery secretion (parotid).

Striated Ducts
* Columnar cells
* Centrally located nucleus
* Eosinophilic cytoplasm
* Prominenty striations
o Indentations of the cytoplasmic membrane with many mitochondria present between the folds
* Some RER and some Golgi, short microvilli
* Modify the secretion
o Hypotonic solution=low sodium and chloride and high potassium
* Basal cells

Terminal excretory ducts
* Near the striated ducts they have the same histology as the striated ducts
* As the duct reaches the oral mucosa the lining becomes stratified
* Goblet cells, basal cells, clear cells.
* Alter the electrolyte concentration and add mucoid substance.

Ductal modification
* Autonomic nervous system
* Striated and terminal ducts
* Modofication via reabsorption and secretion of electrolytes
* Final product is hypotonic
* Rate of salivary flow
o High: Sodium and chlorine up; potassium down

Connective tissue
* Fibroblasts
* Inflammatory cells
* Mast cells
* Adipose cells
* Extracellular matrix
o Glycoproteins and proteoglycans
* Collagen and oxytalan fibers
* Blood supply

Nerve supply
* No direct inhibitory innervation
* Parasympathetic and sympathetic impulses
* Parasympathetic are more prevalent.
* Parasympathetic impulses may occur in isolation, evoke most of the fluid to be excreted, cause exocytosis, induce contraction of myoepithelial cells (sympathetic too) and cause vasodilatation.
* There are two types of innervation: Epilemmal and hypolemmal
* beta-adrenergic receptors that induce protein secretion
* L-adrenergic and cholinergic receptors that induce water and electrolyte secretion

Hormones can influence the function of the salivary glands. They modify the salivary content but cannot iniate salivary flow.

Age changes
* Fibrosis and fatty degenerative changes
* Presence of oncocytes (eosinophilic cells containing many mitochondria)

Clinical Considerations
* Obstruction
* Role of drugs
* Systemic disorders
* Bacterial or viral infections
* Therapeutic radiation
* Formation of plaque and calculus

Salivary Glands.ppt


24 March 2010

Examination of Urine

Examination of Urine
By:Terry Kotrla, MS, MT(ASCP)
Austin Community College

Urine Color

* Normal urine color ranges from pale yellow to deep amber — the result of a pigment called urochrome
o B vitamins turn urine an eye-popping neon yellow BUT may also indicate liver disease.
o porphyria, a disease that affects your skin and nervous system, turns urine the color of port wine.

Urine Color

* Most changes in urine color are harmless and temporary and may be due to:
o Certain foods – beets may turn urine red
o Dyes in foods/drinks
o Supplements – vitamins
o Prescription drugs
* Unusual urine color can indicate an infection or serious illness .

Suggested Colors

* pale yellow (straw)
* light yellow
* yellow
* green-yellow (olive)
* red-yellow
* red
* red-brown
* brown-black
* black
* milky

Examples of Urine Color

Urine Clarity

* During the visual inspection, the MLT observes the urine's and determines how clear it is (its clarity).
* Urine clarity refers to how clear the urine is.
* Terms used: clear, slightly cloudy, cloudy, or turbid.
* “Normal” urine can be clear or cloudy.
* The clarity of the urine is not as important as the substance that is causing the urine to be cloudy.

Urine Clarity

* Substances that cause cloudiness but that are not considered unhealthy include:
o mucous,
o sperm and prostatic fluid,
o cells from the skin,
o normal urine crystals, and
o contaminants (like body lotions and powders).
* Other substances that can make urine cloudy (such as red blood cells, white blood cells, or bacteria) indicate a condition that requires attention.

Examples of Urine Clarity

Urine Color and Clarity

* Urine color and clarity can indicate what substances may be present in urine.
* Confirmation of suspected substances is obtained during the chemical and microsopic examination.

Chemical Examination

* Reagent strips are used only once and discarded.
* Testing
o Perform within 1 hour after collection
o Allow refrigerated specimens to return to room temperature.
o Dip strip in fresh urine and compare color of pads to the color chart after appropriate time period.
o Instruments are available which detect color changes electronically

Using Reagent Strips

* BRIEFLY dip the strip in urine.
* Colors are matched to those on the bottle label at the appropriate times.
* Timing is critical for accurate results.

Reagent Strips


* Presence of glucose (glycosuria) indicates that the blood glucose level has exceeded the renal threshold.
* Useful to screen for diabetes.


* Bilirubin is a byproduct of the breakdown of hemoglobin.
* Normally contains no bilirubin.
* Presence may be an indication of liver disease, bile duct obstruction or hepatitis.
* Since the bilirubin in samples is sensitive to light, exposure of the urine samples to light for a long period of time may result in a false negative test result.


* Ketones are excreted when the body metabolizes fats incompletely (ketonuria)

Specific Gravity

* Specific gravity reflects kidney's ability to concentrate.
* Want concentrated urine for accurate testing, best is first morning sample.
* Low – specimen not concentrated, kidney disease.
* High – first morning, certain drugs


* Presence of blood may indicate infection, trauma to the urinary tract or bleeding in the kidneys.
* False positive readings most often due to contamination with menstrual blood.


* pH measures degree of acidity or alkalinity of urine


* Presence of protein (proteinuria) is an important indicator of renal disease.
* False negatives can occur in alkaline or dilute urine or when primary protein is not albumin.


* Urobilinogen is a degradation product of bilirubin formed by intestinal bacteria.
* It may be increased in hepatic disease or hemolytic disease


* Nitrite formed by gram negative bacteria converting urinary nitrate to nitrite


* Leukocytes (white blood cells) usually indicate infection.
* Leucocyte esterase activity is due to presence of WBCs in urine while nitrites strongly suggest bacteriuria.

Normal Values

* Negative results for glucose, ketones, bilirubin, nitrites, leukocyte esterase and blood.
* Protein negative or trace.
* pH 5.5-8.0
* Urobilinogen 0.2-1.0 Ehrlich units

Handling and Storage of Strips

* Handling and Storage
o Keep strips in original container
o Do not touch reagent pad areas
o Reagents and strips must be stored properly to retain activity
+ Protect from moisture and volatile fumes
+ Stored at room temperature
o Use before expiration date


* Dip strip briefly, but completely into well mixed, room temperature urine sample.
* Withdraw strip.
* Blot briefly on its side.
* Keep the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.

Sources of Error

* Timing - Failure to observe color changes at appropriate time intervals may cause inaccurate results.
* Lighting - Observe color changes and color charts under good lighting.
* QC - Reagent strips should be tested with positive controls on each day of use to ensure proper reactivity.
* Sample - Proper collection and storage of urine is necessary to insure preservation of chemical.

Sources of Error

* Testing cold specimens - would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing
* Inadequate mixing of specimen - could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping
* Over-dipping of reagent strip - will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine

Examination of Urine


Blood Collection

Blood Collection

An overview of the process involved in collecting donor blood

Donor Screening
* Starts with the donor and first impressions are critical
* Clean, well lit donation facility from waiting room to collection area
* Pleasant, professional staff who can ask the appropriate questions, observe and interpret the responses, and ensure that the collection process is as pleasant as possible

Blood Bank versus Blood Center
* Confusion exists and terms are sometimes used inappropriately
* Blood bank in a hospital is also known as the transfusion service, performs compatibility testing and prepares components for transfusion
* Blood Center is the donation center, screens donors, draws donors, performs testing on the donor blood, and delivers appropriate components to the hospital blood bank

Standards, Regulations, Governing Bodies
* Strict guidelines exist and inspections are performed in both blood centers and blood banks to ensure the safety of the donors and patients
* Some or all of the following agencies may be involved:
o AABB – American Association of Blood Banks
o FDA – Food and Drug Administration
o CAP – College of the American Pathologists
o JCAHO - Joint Commission on the Accreditation of Hospital Organizations
o NCCLS – National Committee for Clinical Laboratory Standards

Donor Screening
* Medical History based on a standardized questionnaire obtains critical information about the donor’s health and risk factors which may make it unsafe for donation
* Physical Exam which includes blood pressure, temperature, pulse and screen for anemia are performed to ensure donor is healthy enough to donate.
* Two goals of screening
o Protect the health of the potential donor
o Protect the health of the potential recipient

Donor Registration
* Donor signs in
* Written materials are given to the donor which explains high risk activities which may make the donor ineligible
* Donor must be informed and give consent that blood will be used for others unless they are in a special donor category
* First time donors must provide proof of identification such as SS#, DL#, DOB, address and any other unique information.
* Repeat donors may be required to show DL or some other photo ID

Frequency of donation
* Whole blood or red blood cells 8 weeks
* Plateletpheresis – up to 24 times/year
* Plasmapheresis– once every 4 weeks, can be done twice a week
* Granulocytes

Medical History
* A thorough history is obtained each time
* Standardized universal questionnaire is used
* Questions are asked that are very intimate in nature but are critical in assessing HIV or HBV risks
* Medications the donor taking are present in plasma, may cause deferral
* Infections the donor has may be passed to recipient, may be cause for deferral

12 Month Deferral
* Any intimate sexual relations with HIV positive, HBV positive, hemophiliacs, drug users or individuals receiving drugs/money for sex.
* Recipient of blood, components or blood products such as coagulation factors
* Sexually transmitted disease-if acquired indicates safe sex not practiced and donor at risk for HIV and HBV
* Travel to malarial endemic country

Temporary Deferrals
* Certain immunizations
o 2 weeks -MMR, yellow fever, oral polio, typhoid
o 4 weeks -Rubella, Chicken Pox
o 2 months – small pox
* Pregnancy – 6 weeks upon conclusion
* Certain medications
o Proscar/Propecia, Accutain – 1 month
o Avodart – 6 months
o Soriatane – 3 years
o Tegison - permanent

Permanent Deferrals
* HIV, HBV, or HCV positive
* Protozoan diseases such as Chagas disease or Babesiosis
* Received human pituitary growth hormone
* Donated only unit of blood in which a recipient contracted HIV or HBV
* Was the only common donor in 2 cases of post-transfusion HIV or HBV in recipient
* Lived in a country where Creutzfeld-Jacob disease is prevalent
* Most cancers except minor skin cancer and carcinoma in-situ of the cervix
* Severe heart disease, liver disease

Helpful Hint
* Permanent deferral – any member of high risk group such as: HIV/HBV/HCV pos, drugs/sex for money, cancer, serious illness or disease, CJD, Chagas disease, Babesiosis
* 12 month deferral – sex with any high risk group, any blood exposure, recipient of blood/blood products, STD, jail/prison, rabies vaccine after exposure, HBIG, malaria
* Have to memorize: medications and vaccinations

* Two stickers
o “Yes, use my blood”
o “No, do not use my blood”
* After interview the donor will place the appropriate bar coded label on the donation record
* If “no” selected the unit is collected, fully tested, but not used for transfusion
* Allows donors who know they are at risk to “save face” if pressured to donate by friends and family

Donor Categories
* “Allogeneic”, “homologous” and “random donor” terms used for blood donated by individuals for anyone’s use
* Autologous – donate blood for your own use only
* Recipient Specific Directed donation – donor called in because blood/blood product is needed for a specific patient
* Directed Donor – patient selects their own donors
* Therapeutic bleeding – blood removed for medical purposes such as in polycythemia vera. NOT used for transfusion.

Auto/Directed Blood Labels
Donor Categories
* Safest is autologous, blood is your own, no risk of disease acquisition
* Most dangerous is Directed Donor, you select a donor who may, unknown to you, be in a high risk category but feels obligated to follow through and donate

Blood Collection
* Materials used are sterile and single use.
* Most important step is preparing the site to a state of almost surgical cleanliness.
* Bacteria on skin, if present, may grow well in stored donor blood and cause a fatal sepsis in recipient
* Use 16-17 gauge needle to collect blood from a single venipuncture within 15 minutes
* Collect 450 +/- 45 mLs of blood

Donor Reactions
* Syncope (fainting)
o Remove needle immediately
* Hyperventilation
o Have donor rebreathe into paper bag.
* Nausea/vomiting
* Twitching/muscle spasms
* Hematoma
* Convulsions – rare, get immediate assistance
* Cardiac difficulties

Post-Phlebotomy Care
* Donor applies pressure for 5 minutes
* Check and bandage site
* Have donor sit up for few minutes
* Have donor report to refreshment area for additional 15 minutes of monitoring

Post-Phlebotomy Instructions
* Eat/drink before leaving
* Wait until staff releases you
* Drink more fluids next 4 hours
* No alcohol until after eating
* Refrain from smoking for 1 hour
* If bleeding continues apply pressure and raise arm
* Faint or dizzy sit with head between knees
* Abnormal symptoms persist contact blood center.
* Remove bandage

Testing Donor Blood
* CANNOT rely on previous testing
* Records must be kept for 5 years

Serological Testing
* ABO/D typing
* Antibody Screen – if positive, ID antibody, cannot make plasma products
* Antibodies to other blood group antigens which are present in the donor may react with recipient red cells resulting in a reaction.

Disease Testing
* Disease testing include:
o HBc
o HIV 1&2
o NAT for HIV-1, HCV & WNV

Results of Testing
* Tests for disease markers must be negative or within normal limits.
* Donor blood which falls outside these parameters must be quarrantined.
* Repeat testing, if still abnormal must dispose.

Transfusion Service Testing
* The only repeat testing required is:
o ABO on red cell products
o D typing (IS) on D negative red cell products
* Plasma products (FFP, CRYO, PLTS) do not require any testing.
* Donor samples must be stored at 1-6C for at least 7 days after transfusion
o ADSOL unit transfused today must save sprig for one week
o Many facilities will pull a sprig from each donor during processing and save all sprigs for 49 days, regardless of expiration of unit

* Blood collection starts with screening of the donor to:
o Ensure they are healthy enough to donate
o Ensure they do not have transmissible diseases
* Many organizations set standards and monitor all aspects of blood collection and administration.
* Collection of blood must be done in such a manner as to ensure sterility of the component.
* Testing of donor blood includes serological testing for ABO/D typing, antibody screening, and testing for markers indicating infection.
* The blood supply is NOT safe, only careful screening and testing can prevent, as much as possible, disease transmission.

Blood Collection


Assessment of Protein Status

Assessment of Protein Status
FCSN 442 - Nutrition Assessment Laboratory
By:Dr. David L. Gee
Central Washington University

Assessment of Protein Status
* Anthropometric Assessment
o body composition estimations
o midarm muscle circumference/area
* Laboratory Assessment
o serum albumin
o other serum proteins (transferrin, prealbumin, retinol-binding protein)
o urinary creatinine excretion
o total lymphocyte count

Midarm Muscle Area
* Estimate of MAMA is an estimate of overall muscle mass
* Assumptions

Midarm Muscle Circumference
* MAMC = AC - (.314 x TSF)

* “…change in arm muscle area is greater than the change in mid-arm circumference. Consequently, changes in upper-arm musculature are not as easily detected by measurement of mid-arm circumference as by AMA. Therefore, AMA is the preferred nutritional index.”

Arm Muscle Area
* AMA = ((MAC - (3.14 x TSF)2 ) / (4 x 3.14)
* adjusted AMA

Guidelines for Interpreting Percentile Values for Arm Muscle Area (appendix R)
Biochemical Assessment of Protein Status
* Two protein compartment model
* “No single test or group of tests can be recommended at this time as a routine and reliable indicator of protein status.” Young, 1990
* “…a combination of measures can produce a more complete picture of protein status.”

Serum Albumin
* Major serum protein
* Most common indicator of depleted protein status
* Half life = 14-20 days
* poor indicator of early protein depletion and repletion
* Levels affected by rate of synthesis (liver disease may reduce levels)
* May reflect level of physiological stress
* Levels affected by abnormal losses
* Levels affected by fluid status
* Normal values: 4.5 g/dL + 35-50 (SD)

Serum Transferrin
* Function: transport protein for iron
* half-life = 8-9 days
* Influenced by other factors
* limited usefulness in protein status assess.

Serum Prealbumin
* aka. transthyretin and thyroxine-binding prealbumin
* functions:
* short half life (2-3d), small body pool
* Returns to normal at beginning of nutritional therapy
* Influenced by other factors
* generally considered preferable than albumin and transferrin

Retinol Binding Protein
* Function: carrier for retinol
* responds like prealbumin
* very rapid turnover (12 hours), very small body pool
* generally not considered to be more useful than prealbumin

* Immune system affected by nutritional status
* Tests of immunocompetence useful functional indicators of nutritional status
* Delayed Cutaneous Hypersensitivty (DCH)
o intradermal injection of antigens
* Total Lymphocyte Count (TLC)

Total Lympocyte Count
* White blood cell count
* TLC = (%lymp x WBC)x100
* Normal = 1200-1800 cells/mm3
* Moderate PCM = 800-1200
* Severe PCM = < 800 Urinary Creatinine Excretion * Creatinine excreted in proportion to muscle mass * LBM estimated by comparing 24-hr urine creatinine excretion with standard based on stature or reference values of 23 and 18 mg/kg for M and F Example: Creatinine Height Index * CHI = (24 hr urine creatinine x 100) / (expected 24 hr urine creatinine for height) o CHI = 1436/1596 x 100 = 90% * expected values in table 9-1 (p306) o CHI > 80% = normal
o CHI = 60-80% = mild protein depletion
o CHI = 40-60% = moderate depletion
o CHI < 40% = severe depletion Assessment of Protein Status


Immune System Overview Mechanisms of Immunosuppression

Immune System Overview Mechanisms of Immunosuppression
By:Bob Luebke
Immunotoxicology Branch
Experimental Toxicology Division

Role of the Immune System in Homeostasis

* Bidirectional interaction with other systems
o Reproduction
o Endocrine

Basics of Immunology
The Immune Response
Innate Immunity
Adaptive (Acquired) Immunity
-Phylogenetically ancient
-Limited recognition
-Rapid (minutes – hours)
- No cell proliferation required
-Limited memory (? mammals)
-First appeared in jawed fishes
- Infinite array of specificities
- Slow (days)
-Requires proliferation and differentiation
-Long-lasting memory

Basics of Immunology
* The adaptive immune response to antigen
Organs of the Immune System
Immune System Anatomy
Organs of the Immune System
Thymus: source of naive T cells
Fate of T Cells in the Thymus
Positive selection: optimal binding to self Ag prevents apoptosis
Negative selection: superoptimal binding to self Ag induces apoptosis
B cells: Tolerance to “Self”
Anergy: low expression of
IgM on surface; can’t bind Ag
Clonal ignorance: too few
copies of Ag in the periphery

Thymus size and architecture:
* May be very sensitive to xenobiotics
* Also sensitive to acute toxicity

Methods for Assessing Direct Immunotoxicity Associated with Exposure to Chemicals
Organs of the Immune System
Spleen: Antigen trapping and presentation, clonal expansion, cellular export
Organs of the Immune System
Lymph nodes: Antigen trapping and presentation, clonal expansion, cellular export
Cells of the Immune System
Innate Immune System: Granulocytes
Neutrophil (“PMN”)
* First responders
* Phagocytosis and killing of bacteria
* Inflammation

* Allergy
* Killing parasite larvae
* Circulating mast cells
* Allergy/anaphylaxis

Innate Immune System: Granulocytes
Neutrophil (“PMN”)
* First responders
* Phagocytosis and killing of bacteria
* Inflammation
Cells of the Immune System
Innate Immune System: Monocytes
Macrophage with ingested
asbestos fiber (encarta.msn.com)
* Phagocytosis and killing of bacteria
* Antigen processing
* Inflammation
Adaptive Immune System: Lymphocytes
Activated B cell
Peripheral blood
Activated T cell (SEM)
* B cells: Mature into plasma cells, secrete antibody (IgM, IgG, IgA, IgE, IgD)
* T cells: T helper - produce stimulatory and regulatory cytokines
* T cells: T cytotoxic/suppressor – contact-dependent cytotoxicity,

regulation of immune response
* NK cells: direct killing of cells (innate arm of IS)

Plasma Cells Produce Antibodies
* IgM: Primary response, efficient agglutination
* IgG: Recall response, highest concentration
* IgA: Mucosal surfaces, trapping of microbes
* IgE: Allery/anaphylaxis

Factors Affecting Immunocompetence
* Age
* Gender
* Genotype
* Nutritional status
* Life style choices
* Acute toxicity
CONCEPT: Individual immunocompetence, in the absence of xenobiotic exposure, is complex, dynamic and affected by fixed and variable factors. At the population level, the “normal” range is broad.

Immunocompetence in the Young: Innate immunity
* Neutrophils
* NK cells
Immunocompetence in the Young: Adaptive immunity
* Humoral immunity
* Cellular immunity
* Resistance to infection
Advanced Age and Immunocompetence

* Innate Immunity
* Adaptive immunity
* Resistance to infection

Gender and Immunocompetence
Genotype and Immunocompetence
Lifestyle and Immunocompetence
* Recreational drug use
* Excessive use of alcohol
* Smoking
* Stress

Xenobiotic Exposure and Immunocompetence

Consequences of Xenobiotic Exposure on Immunocompetence
“Pre-immune” Mechanisms of Defense
Immune Mediated Resistance to Infection
Organism Factors Influencing Host Resistance
Mechanisms of Chemically-induced Immunosuppression
Mechanisms of Suppression:
Effects on Supply of Cells
Mechanisms of Suppression: Effects on Supply of Cells
UVB (320-280 nm) exposure
Mechanisms of Suppression: Tolerance Induction (and then some)
Mechanisms of Suppression: Tolerance Induction (human studies)
Mechanisms of Suppression: Modulation of cytokine production
Mechanisms of Suppression: Th1/Th2 Polarization
Mechanisms of Suppression: Disruption of innate immunity
Human and Mouse Macrophage Responses to Ozone in vivo
Mechanisms of Chemically-induced Immunosuppression
Mechanisms of Suppression:
* Reduced supply of immune system cells
* Misdirection of the immune system
* Direct effects on cells
* Combination of effects
Decreased Host Resistance: Implications for Human Health

Immune System Overview Mechanisms of Immunosuppression


23 March 2010

Metabolic Disorders - Inborn Errors of Metabolism

Metabolic Disorders - Inborn Errors of Metabolism
By:Dr. Sara Mitchell

* Proteins - what are they and what do they do?
* Amino Acids - what are they and what do they do?

Eight Essential Amino Acids
* Tryptophan
* Lysine
* Methionine
* Phenylaline
* Theronine
* Valine
* Leucine
* Isolecucine

Inborn Errors of metabolism
* Affects amino acid & protein, carbohydrate, and lipid metabolism.
* Most disorders are autosomal recessive in transmission
* Most disorders are evident at or soon after birth.
* Early detection and treatment are essential to the prevention of irreversible cognitive impairment and early death

Newborn Screening: What is it?
* A test developed in 1961 by Dr. Robert Guthrie to evaluate infants for certain genetic anomalies, inborn errors of metabolism, and other disorders.


Phenylketonuria (PKU):What is it?
* The most common amino acidemia. Classic PKU develops in the absence of the enzyme phenylalanine hydroxylase.
* Incidence

Phenylketonuria: How’s it happen?
* Cause
o absent Phenylalanine hydroxylase causes a build up phenylalanine
* Effect

* Treatment
* Prognosis

Galactocemia: What is it?
* An inborn error of carbohydrate metabolism in which the hepatic enzyme galactose 1-phosphate uridine transferase is absent.
* Incidence

Galactocemia: How does it happen?
Galactocemia: What are the clinical manifestations?
Galactocemia: Diagnosis & Treatment
* Diagnosis
* Treatment

Metabolic Disorders - Inborn Errors of Metabolism.ppt


Amino Acid Catabolism: Carbon Skeletons

Amino Acid Catabolism: Carbon Skeletons
Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.

Molecular Biochemistry II

Amino Acid Carbon Skeletons
Amino acids, when deaminated, yield a-keto acids that, directly or via additional reactions, feed into major metabolic pathways (e.g., Krebs Cycle).
Amino acids are grouped into 2 classes, based on whether or not their carbon skeletons can be converted to glucose:

o glucogenic
o ketogenic.

Carbon skeletons of glucogenic amino acids are degraded to:
o pyruvate, or
o a 4-C or 5-C intermediate of Krebs Cycle. These are precursors for gluconeogenesis.
Glucogenic amino acids are the major carbon source for gluconeogenesis when glucose levels are low.
They can also be catabolized for energy, or converted to glycogen or fatty acids for energy storage.

Carbon skeletons of ketogenic amino acids are degraded to:
o acetyl-CoA, or
o acetoacetate.

Acetyl CoA, & its precursor acetoacetate, cannot yield net production of oxaloacetate, the gluconeogenesis precursor.
For every 2-C acetyl residue entering Krebs Cycle, 2 C leave as CO2.
Carbon skeletons of ketogenic amino acids can be catabolized for energy in Krebs Cycle, or converted to ketone bodies or fatty acids.
They cannot be converted to glucose.
The 3-C a-keto acid pyruvate is produced from alanine, cysteine, glycine, serine, & threonine.
Alanine deamination via Transaminase directly yields pyruvate.
Serine is deaminated to pyruvate via Serine Dehydratase.
Glycine, which is also product of threonine catabolism, is converted to serine by a reaction involving tetrahydrofolate (to be discussed later).

The 4-C Krebs Cycle intermediate oxaloacetate is produced from aspartate & asparagine.
Aspartate transamination yields oxaloacetate.
Aspartate is also converted to fumarate in Urea Cycle. Fumarate is converted to oxaloacetate in Krebs cycle.
Asparagine loses the amino group from its R-group by hydrolysis catalyzed by Asparaginase.
This yields aspartate, which can be converted to oxaloacetate, e.g., by transamination.
The 4-C Krebs Cycle intermediate succinyl-CoA is produced from isoleucine, valine, & methionine.
Propionyl-CoA, an intermediate on these pathways, is also a product of b-oxidation of fatty acids with an odd number of C atoms.
The branched chain amino acids initially share in part a common pathway.
Branched Chain a-Keto Acid Dehydrogenase (BCKDH) is a multi-subunit complex homologous to Pyruvate Dehydrogenase complex.
Genetic deficiency of BCKDH is called Maple Syrup Urine Disease (MSUD).
High concentrations of branched chain keto acids in urine give it a characteristic odor.
Propionyl-CoA is carboxylated to methylmalonyl-CoA.
A racemase yields the L-isomer essential to the subsequent reaction.
Methylmalonyl-CoA Mutase catalyzes a molecular rearrangement: the branched C chain of methylmalonyl-CoA is converted to the linear C chain of succinyl-CoA.
The carboxyl that is in ester linkage to the thiol of coenzyme A is shifted to an adjacent carbon atom, with opposite shift of a hydrogen atom.

Recall that coenzyme A is a large molecule.
Coenzyme B12, a derivative of vitamin B12 (cobalamin), is the prosthetic group of Methylmalonyl-CoA Mutase.
A crystal structure of the enzyme-bound coenzyme B12.
Coenzyme B12 contains a heme-like corrin ring with a cobalt ion coordinated to 4 ring N atoms.
o methyl C atom of 5'-deoxyadenosine (not shown).
o an enzyme histidine N
When B12 is free in solution, a ring N of the dimethylbenzimidazole serves as axial ligand to the cobalt.
When B12 is enzyme-bound, a His side-chain N substitutes for the dimethylbenzimidazole.
Within the active site, the Co atom of coenzyme B12 has 2 axial ligands:
Homolytic cleavage of the deoxyadenosyl C-Co bond during catalysis yields a deoxyadenosyl carbon radical, as Co3+ becomes Co2+.
Reaction of this with methylmalonyl-CoA generates a radical substrate intermediate and 5'-deoxyadenosine.
Following rearrangement of the substrate, the product radical abstracts a H atom from the methyl group of 5'-deoxyadenosine.
This yields succinyl-CoA and the 5'-deoxyadenosyl radical, which reacts with coenzyme B12 to reestablish the deoxyadenosyl C-Co bond.

Methyl group transfers are also carried out by B12 (cobalamin).
Methyl-B12 (methylcobalamin), with a methyl axial ligand substituting for the deoxyadenosyl moiety of coenzyme B12, is an intermediate of such transfers.
E.g., B12 is a prosthetic group of the mammalian enzyme that catalyzes methylation of homocysteine to form methionine (to be discussed later).
o Vitamin B12 is synthesized only by bacteria.
Ruminants get B12 from bacteria in their digestive system.
Humans obtain B12 from meat or dairy products.
o Vitamin B12 bound to the protein gastric intrinsic factor is absorbed by cells in the upper part of the human small intestine via receptor-mediated endocytosis.
B12 synthesized by bacteria in the large intestine is unavailable.
Strict vegetarians eventually become deficient in B12 unless they consume it in pill form.
o Vitamin B12 is transported in the blood bound to the protein transcobalamin, which is recognized by a receptor that mediates uptake into body cells.

Explore via Chime
Methylmalonyl-CoA Mutase
with its prosthetic group,
Coenzyme B12.
Desulfo-CoA (without the
thiol) is at the active site.
The deoxyadenosyl moiety is lacking in the crystal.

The 5-C Krebs Cycle intermediate a-ketoglutarate is produced from arginine, glutamate, glutamine, histidine, & proline.
Glutamate deamination via Transaminase directly yields a-ketoglutarate.
Glutamate deamination by Glutamate Dehydrogenase also directly yields a-ketoglutarate.
Histidine is first converted to glutamate. The last step in this pathway involves the cofactor tetrahydrofolate.
Tetrahydrofolate (THF), which has a pteridine ring, is a reduced form of the B vitamin folate.
Within a cell, THF has an attached chain of several glutamate residues, linked to one another by isopeptide bonds involving the R-group carboxyl.
THF exists in various forms, with single-C units, of varying oxidation state, bonded at N5 or N10, or bridging between them.
In these diagrams N10 with R is r-aminobenzoic acid, linked to a chain of glutamate residues.
The cellular pool of THF includes various forms, produced and utilized in different reactions.
N5-formimino-THF is involved in the pathway for degradation of histidine.
Reactions using THF as donor of a single-C unit include synthesis of thymidylate, methionine, f-methionine-tRNA, etc.
In the pathway of histidine degradation, N-formiminoglutamate is converted to glutamate by transfer of the formimino group to THF, yielding N5-formimino-THF.
Because of the essential roles of THF as acceptor and donor of single carbon units, dietary deficiency of folate, genetic deficiencies in folate metabolism or transport, and the increased catabolism of folate seen in some disease states, result in various metabolic effects leading to increased risk of developmental defects, cardiovascular disease, and cancer.

Aromatic Amino Acids
Aromatic amino acids phenylalanine & tyrosine are catabolized to fumarate and acetoacetate.
Hydroxylation of phenylalanine to form tyrosine involves the reductant tetrahydrobiopterin. Biopterin, like folate, has a pteridine ring.
Dihydrobiopterin is reduced to tetrahydrobiopterin by electron transfer from NADH.
Thus NADH is secondarily the e- donor for conversion of phenylalanine to tyrosine.
Overall the reaction is considered a mixed function oxidation, because one O atom of O2 is reduced to water while the other is incorporated into the amino acid product.
O2, tetrahydrobiopterin, and the iron atom in the ferrous (Fe++) oxidation state participate in the hydroxylation.
O2 is thought to react initially with the tetrahydrobiopterin to form a peroxy intermediate.
Phenylalanine Hydroxylase includes a non-heme iron atom at its active site.
X-ray crystallography has shown the following are ligands to the iron atom:
His N, Glu O & water O.
(Fe shown in spacefill & ligands in ball & stick).
deamination via transaminase) accumulate in blood & urine.
Mental retardation results unless treatment begins immediately after birth. Treatment consists of limiting phenylalanine intake to levels barely adequate to support growth. Tyrosine, an essential nutrient for individuals with phenylketonuria, must be supplied in the diet.
Genetic deficiency of Phenylalanine Hydroxylase leads to the disease phenylketonuria.
Phenylalanine & phenylpyruvate (the product of phenylalanine
Tyrosine is a precursor for synthesis of melanins and of epinephrine and norepinephrine.
High [phenylalanine] inhibits Tyrosine Hydroxylase, on the pathway for synthesis of the pigment melanin from tyrosine. Individuals with phenylketonuria have light skin & hair color.
Methionine S-Adenosylmethionine by ATP-dependent reaction.
SAM is a methyl group donor in synthetic reactions.
The resulting S-adenosylhomocysteine is hydrolyzed to homocysteine.
Homocysteine may be catabolized via a complex pathway to cysteine & succinyl-CoA.
Or methionine may be regenerated from homocysteine by methyl transfer from N5-methyl-tetrahydrofolate, via a methyltransferase enzyme that uses B12 as prosthetic group.
The methyl group is transferred from THF to B12 to homocysteine.
Another pathway converts homocysteine to glutathione.
In various reactions, S-adenosylmethionine (SAM) is a donor of diverse chemical groups including methylene, amino, ribosyl and aminoalkyl groups, and a source of 5'-deoxyadenosyl radicals.
But SAM is best known as a methyl group donor.

S-adenosylmethionine as methyl group donor
o methylation of bases in tRNA
o methylation of cytosine residues in DNA
o methylation of norepinephrine epinephrine
o conversion of the glycerophospholipid
phosphatidyl ethanolamine phosphatidylcholine via methyl transfer from SAM.
Enzymes involved in formation and utilization of S-adenosylmethionine are particularly active in liver.
Liver has important roles in synthetic pathways involving methylation reactions, & in regulation of blood methionine.
Methyl Group Donors

Methyl group donors in synthetic reactions include:

* methyl-B12
* S-adenosylmethionine (SAM)
* N5-methyl-tetrahydrofolate (N5-methyl-THF)
Lysine & Tryptophan

Amino Acid Catabolism: Carbon Skeletons.ppt


Phenylketonuria (PKU)

Phenylketonuria (PKU)
By:Ashley Ryan

What is PKU?

* An inherited metabolic disease in which mental retardation can be prevented by a specific diet
* 1 out of 50 people are carriers of defective gene; 1 in 10,000 births
* Rare condition where a baby is born lacking the ability to break down phenylalanine.
* Phenylalanine is an amino acid found in many foods. It is characterized by higher than normal levels of phenylalanine in the blood which can cause damage to the brain and mental retardation
* The brain suffers and is damaged due to a tremendous buildup of phenylalanine

*This then results in damage of the CNS & causes brain damage

Causes & Symptoms
* Since PKU is inherited it is passed down through families
* Which means both parents must pass on the defective gene to there offspring; known as an autosomal recessive trait
* Phenylalanine is involved in the body’s production of melanin which is the pigment for skin and hair color – children with PKU generally have lighter skin, hair and eyes
* Other symptoms include

* PKU is in fact treatable with the correct strictly followed diet very low in phenylalanine
* If diet is not followed brain impairment can occur or error of metabolism can be associated with M.R in first year of life.
* Association with attention-deficit hyperactivity disorder (ADHD) most common problem in those who don’t follow a strict diet
* If diet is properly followed esp. in first few years of life where it is most crucial an outcome of better physical and mental health will follow
* Examples of foods low in phenylalanine: milk, eggs, fish oil, special formula called Lofenalac .
* Lofenalac provides essential amino acids and can be used throughout life. It not only provides amino acids but also vitamins and minerals.
* Can think of it as a super food for PKU patients

Testing for PKU
* It is IMPERTATIVE that phenylalanine restrictions on diet is introduced after birth to prevent the neurodevelopment effects of PKU
* How is PKU tested?
* Blood is routinely drawn from the infants for testing
* A “heel stick” is done and then collected on special blotter paper
* Routine testing includes phenylketonuria and blood type

* Overall highly recommend to have strong relationship with physician
* An Enzyme Assay can determine if parents carry defective gene
* Chorionic villus Sampling - screen unborn baby for possibility of PKU
* It is very important that women with PKU closely follow a strict low-phenylalanine diet both before becoming pregnant and throughout the pregnancy, since build-up of this substance will damage the developing baby even if the child has not inherited the defective gene.

Age and Diet- controversy
* The age when a diet can or should be discontinued has been debatable over decades
* Generally- PKU centers advise a life-long diet  especially for female patients
* A study was done that looked at progress of children who ended their diet at an early age
* the main focus of the study was the effects on neurological/ intellectual performance
* The participants abilities were compared during treatment and after the diet was discontinued
* RESULTS- It was shown that children who maintained the diet had fewer deficits to those terminating the diet before the age of 10
* Overall the study said a diet should remain strict to at least the age of 10!
* Although this was said also recommended to maintain diet in adulthood
* can be modified but not completely eliminated

Issues in Adults with PKU
* Several studies said that discontinuation of diet effect
* New problems with PKU:
* Adults w/ PKU who remained on diet but weren't as strict w what they ate showed white matter abnormalities when given MRI indicating a reduction in myelin.
* *** These conditions disappeared after reintroducing the strict diet ***
* Neurological investigations in early treated adults w/ PKU who stopped the diet showed higher incidence of neurological signs including:

-clumsy motor coordination
* Investigation on Psychological problems also
-severe behavior/ psychiatric problems are seen in profound retarded/untreated adults w/ PKU in their 30’s-40’s.
* Claims that reintroduction of restricted diet symptoms can sometimes be reversible
* Adults who discontinued the diet have had cases of
* - depression, anxiety, social withdrawal, phobias, low self-esteem, neurotic behavior
* In 2009 it was stated PKU patients should be encouraged to remain on a life long diet and also recommended to:

-take nutritional supplements
* Blood PhE levels should be monitored every 3 months
* Yearly clinical review
* PKU pregnant women recommendations include:
* Being under control of physician specialized in
* metabolic disease, gynecologist, and dietician
* Detailed ultrasound @ 20 weeks of gestation
* Seen every 3 to 4 weeks and blood PhE levels monitored at least 1 a week

Factors to consider when people discontinue restricted diets
* Difficulty maintaining diet for older children
* State support of formula costs is decreasing
* In 1978 85% of PKU programs received financial backing, within 6 years 66% of people received financial support
* Formula Cost can range from $5,000 to $7,000 a year. For a young adult and families can be a problem financially if not receiving any support
* Therefore, when people do stop the restricted diet its important to consider and assess financial, nutritional, social, psychological problems that people encounter trying to maintain the diet. And remember that in some cases people don’t discontinue the diet because they want to they may be unable to.

Reference Page
Phenylketonuria (PKU)


Case Study: Phenylketonuria (PKU)

Case Study: Phenylketonuria (PKU)
By: Bobby Orr
Adam Edwards
Danielle Heinbaugh

Introduction: What is PKU?
* PKU (Phenylketonuria) is a disorder defined as the inability to metabolize the essential amino acid phenylalanine
* This can cause mental retardation, if untreated, although sufficient treatment can occur immediately after birth

* The main symptom consists of mild to moderate mental retardation, but this is easily prevented through treatment
* However, other side effects include seizures, vomiting, a “mousy odor”, and behavioral self-mutilation
* In some cases, treatment can reduce or reverse the mental retartadtion

The Guthrie Test:
* determines the phenylalanine level in the blood
* should be done on the second or third day of life
* is a screening test done to identify elevated phenylalanine levels it is not diagnostic
* PKU babies’ phenylalanine level is usually 20-40 mg/dl in comparison with normal levels of 4-6 mg/dl.

How the Guthrie Test works:
* Blood on filter paper is placed on agar plates with a strain of bacillus subtilis that requires phenylalanine for growth.
* The presence of growth is indicated by a halo surrounding the filter paper.
* If positive, blood phenylalanine and tyrosine levels are determined, and if elevated, a confirmatory assay for phenylalanine hydroxylase is done.

PKU Inheritance:
* Inherited as autosomal recessive disorder.
* Variation to classical symptoms is result of compound heterogeneity.
* 65 allelic variants make compound heterogeneity more common then homogeneity for the same allele.

Treatment of PKU:
* Phenylketonuria is treatable with a low phenylalanine diet.
* phenylalanine levels should be kept below 15 mg per deciliter
* Nutra sweet is especially high in phenylalanines

Genetic Counseling:
* Tell the parents that the baby will be normal if they follow the prescribed dietary guidelines
* The child is normally out of danger of the disease after puberty
* Phenylalanine should be avoided
o Stay away from nutra sweet, meats, dairy products

Case Study: Phenylketonuria (PKU)

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