The Team Approach

Routine urine handling requires properly labeled and covered containers but usually doesn't require protection from light. If porphyrinogens or porphyrins are suspected, the specimen should also be protected from light, and communication with nursing staff may be necessary to get a light-protected urine sample.

Case Scenario 5-3 Acute Variegate Porphyria: Darkening Urine Samples in the Laboratory (continued)

TEST METHODOLOGY 5-3. URINE PORPHYRIN, PORPHYRINOGEN, AND PORPHOBILINOGEN (continued)

Watson Schwartz Test

PBG in UBG in aqueous layer butanol layer

Figure 5-8. Watson and Schwarz procedure.

The Reaction: Porphyrinogen

Watson and Schwartz developed a qualitative test used in the detection of porphyrinogen. It uses Ehrlich's reagent, consisting of p-dimethylaminobenzaldehyde. Red color remaining in the aqueous supernatant (upper layer) is usually due to porphobilinogen. To increase specificity, the upper layer is further extracted with butanol. Porphobilinogen is then found in the lower aqueous layer. Any red color in that layer is due to porphobilinogen.

Porphobilinogen + Ehrlich's aldehyde reagent ^ red color

Interferences

Urobilinogen, indole, and indicane can all react pinkish-red. Further extractions can remove compounds such as chloroform, which extracts urobilinogen (UBG) to the lower level, or «-butanol, which extracts UBG to the upper level. Variations to improve specificity and minimize interferences include the addition of sodium acetate to increase pH, buffer, and enhance color. In the primary extraction, chloroform is added to extract UBG to the lower organic layer while porphobilinogen (PBG) stays in the upper aqueous layer. Confirmatory extraction with «-butanol extracts UBG into the upper («-butanol) layer and PBG stays in the lower (urine) layer. For verification, HCl is added to a fresh urine aliquot. If color develops, interfering substances are present.

Case Scenario 5-3 Acute Variegate Porphyria: Darkening Urine Samples in the Laboratory (continued)

TEST METHODOLOGY 5-3. URINE PORPHYRIN, PORPHYRINOGEN, AND PORPHOBILINOGEN (continued)

The Reaction: Porphobilinogen

A new semiquantitative screening test for urinary PBG is available. In this test, the urine is pretreated with ion-exchange resin and the color of the Ehrlich-PBG reaction is matched against a color chart for semiquantitative estimation of concentrations greater than 6 mg/L.13 This test is recommended as a replacement for the Watson and Schwartz test. If positive, more specific tests should then be performed.14

The Specimen

Random urine; protect the specimen from light until analysis. Reference Ranges Negative is the normal result.

Hemoglobin Synthesis in the Context of Porphyrins

As mentioned at the beginning of this chapter, hemoglobin is synthesized from heme and globin. There are four subunits of the hemoglobin molecule: two identical pairs of polypeptide chains (the globins) each with a cleft on the exterior, which contains the heme (iron-porphyrin) group.7 The types of polypeptides are named alpha, beta, delta, gamma, or epsilon.

Normally, the adult has more than 96% of total hemoglobin (Hb) as Hb A. This is composed of four polypeptides that are made of two alpha and two beta chains. There also may be a small percentage of Hb F, composed of two alpha chains and two gamma chains, and a small percentage of Hb A2, composed of two alpha chains and two delta chains. Hemoglobinopathies are diseases that result in structural abnormalities of globin chains, while thalassemias are diseases resulting from decreased synthesis of globin chains. Both types of disorders affect the proportion of normal hemoglobins. A clinical hematology textbook can provide more details about those disorders.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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Responses

  • mandy ebersbach
    What is ubg in urine tests?
    7 years ago
  • donald shaw
    Why butanol is added watsons test?
    1 year ago

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