MEASUREMENT OF HUMAN GROWTH-HORMONE RECEPTOR MESSENGER-RIBONUCLEIC-ACID BY A QUANTITATIVE POLYMERASE CHAIN REACTION-BASED ASSAY - DEMONSTRATION OF REDUCED EXPRESSION AFTER ELECTIVE SURGERY

Studies of GH receptor (GHR) gene expression in human tissues have bee n hampered by the limited amount of tissue available for analysis and the low sensitivity of conventional methods. We have developed a quant itative reverse transcriptase-PCR assay for measurement of GHR messeng er ribonucleic acid levels in small human tissue biopsies. To compensa te for sample to sample variation, an internal RNA standard, which dif fers from the wild-type GHR transcript by only a few nucleotides, was reverse transcribed and amplified together with the GHR transcripts. P CR was carried out using one biotinylated primer to permit the purific ation of single stranded PCR products on streptavidin-coated microtite r plates. The ratio between the wild-type and mutated transcripts was determined by two separate minisequence reactions in which a primer, a nnealed immediately 3' of a variable nucleotide, was extended by a sin gle H-3-labeled nucleotide, complementary to either the wild-type or m utated sequence. The assay range was 0.125-8 x 10(5) transcripts/sampl e, the mean intraassay coefficient of variation was 8.7%, and the lowe r limit of detection was 0.125 x 10(5) transcripts/sample. GHR messeng er ribonucleic acid levels were detectable in small amounts (10-100 ng ) of total RNA extracted from adipose tissue, skeletal muscle, and liv er. The GHR gene expression in liver was approximately 10-fold higher than that in skeletal muscle, whereas intermediate levels were found i n adipose tissue. In nine patients undergoing elective abdominal surge ry, GHR gene expression in skeletal muscle was reduced on day 3 after surgery compared to the baseline level. The decrease in GHR gene expre ssion was accompanied by a decrease in skeletal muscle glutamine. This suggests that the postoperative protein catabolism may be caused at l east partly by acquired GH insensitivity due to reduced expression of the GHR gene.