Forensic Biochem Exam 2

How much saliva is produced daily?

1-1.5 liters

What is saliva made of?

Mostly water

Small quantities of electrolytes, proteins, antibodies, enzymes, oral epithelial cells

What produces saliva?

Salivary glands

◦~70% from submandibular glands

◦~25% from the parotid glands

Confirmatory tests for saliva

None

Presumptive tests for saliva

Based primarily on the identification of:

•Inorganic anions (thiocyanate and nitrate)

•Enzymes (alkaline phosphatase or a-amylase)

Organs of the urinary system

Kidneys, ureter, bladder, urethra

Purpose of urinary system

Eliminates soluble toxic wastes that are cellular metabolic by-products

Urinary system pathway

Formation of urine takes place in the kidneys

Filtered to reabsorb water, glucose, nutrients

Waste (ions, ammonia, urea, etc) secreted through ureter to bladder before its excreted through the urethra

Most abundant waste product

Urea

Inorganic ions in urine

SO42- - barium salt BaCl2(aq) + SO42-  =  BaSO4(s) + 2Cl-

PO43- - ammonium phosphomolybdate (yellow)

Cl-       - AgNO3  (AgCl precipitate)

DMAC

Urine presumptive test

0.05% para dimethyl amino cinnamaldehyde

Detects amines (particularly urea)

    Acetone/HCl

     presence of urine > dark pink/red

Amylose

Type of starch

Straight chained linear polymer of glucose units held in the chain by $a$ 1-4 glycosidiclinkages

Amylopectin

Type of starch

Branched glucose polymer, containing $a$ 1-6  linkages at branch points

$\alpha$-Amylase

In saliva

Hydrolyzes $a$ 1-4 bonds in starch (cannot hydrolyze branched points - $a$ 1-6 bonds)

AMY 1 Form of Amylase

Salivary locus

Perspiration, breast milk

AMY 2 Form of Amylase

Pancreatic locus

Semen, urine, fecal material, vaginal secretions

Forms of Amylase

Two

Sequences are highly homologous

Antibodies will recognize both

Makeup of semen

10-25% Spermatozoa

75-90% Accessory gland secretions (seminal fluid)

Spermatozoa Pathway

Formed in the testes

Transported and stored in the epididymis where they undergo functional maturation

Transferred through vas deferens to the ejaculatory duct

Into the urethra where they are joined with secretions from the prostate

Makeup of Accessory gland secretions

Prostate (~30%)

•High concentrations of acid phosphatase and prostate specific antigen (PSA)

•Used in forensic testing

Seminal vesicles (~60%)

•Secreted proteins play role in coagulation of the ejaculate

•Contains flavin

•Causes semen to fluorescence (UV)

 

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Typical ejaculation

1-6 mL of semen (average ~3 mL)

Volume of ejaculate depends on

Time from previous ejaculate

Metabolic activity of glands (male hormone dependent)

Partial ductal obstruction and retrograde ejaculation

Normospermia

50-150 million sperm/mL (average ~100 million/mL)

Oligospermia/ Aspermia

Oligo = < 20 million/mL

Aspermia = no sperm

Barberio crystal test

Polyamine presumptive test for semen

Spermine phosphate crystals formed by reaction of spermine with anions (phosphate or picrate)

Florence crystal test

Polyamine presumptive test for semen

Insoluble choline periodide crystals when treated with iodine solution

Seminal Acid phosphatase (SAP)

Synthesized in the prostate, present in high levels in semen

Elevated levels of SAP in blood indicative of prostate cancer

Active at acid pH

Not affected by vasectomies

SAP Presumptive Test

Most common screening test

Enzyme which cleaves a variety of organic phosphates: phenyl-, p-nitrophenyl, a-naphthyl, thymolpthalein

Sensitive but not specific. (vaginal acid phosphatase. Slower reaction than SAP)

Prostate Specific Antigen (p30/PSA/KLK3)

Major protein in seminal fluid

Responsible for hydrolyzing semenogelin:

• Creates gel matrix around ejaculated sperm

• After action by p30, gel is broken down allowing sperm to move freely

Important clinical indicator of cancer

Confirmatory test for semen?

• present in urine, peripheral blood, fecal material, sweat and breast milk

P30/PSA Identification methods

Crossover electrophoresis

Ouchterlony double diffusion

Immunochromatography

• 4 ng/ml sensitivity (i.e. 1 in 106 dilution of semen)

• can detect p30 in male urine (260 ng/ml)

ELISA

Confirmatory Sperm Identification (extraction)

1. physical removal by agitation

2. destruction of substrate by acid/base digestion

3. observation while remaining attached to thread

Confirmatory Sperm Identification (visualized)

Directly without staining

Use of histological stains ￿

• nuclear fast (NF) red/picroindigocarmine solution (Christmas Tree)

• hematoxylin/eosin (H & E)

Urine Confirmatory?

Tamm-Horsfall protein (THP)

• most abundant protein in urine

Exclusively synthesized in epithelial cells of Henle’s loop (kidney)

ELISA or Immunochromatographic (RSID-Urine)

Fecal Material Testing

Heme degradation products that are excreted into fecal material

Add zinc acetate in ethanol solution  green fluorescence in UV light

Also reacts with degradation products in urine

AMY 1 inhibited by

wheat lectin

AMY 2 inhibited by

kidney bean

extract