Erythrocyte amino acids in health and renal failure and their association to the IGF-I/IGFBP-1 axis

Abnormalities in amino acids (AA) metabolism in uraemia have mainly been reported to occur in plasma and muscle. No investigation has been undertaken to evaluate the AA levels in plasma, muscle and red blood cells (RBC) simultaneously, although the RBC pool of free AA constitutes a large proportion of the free AA in blood, and RBC are involved in the interorgan transport of AA. Moreover, in most of the earlier studies reporting RBC AA levels in different clinical conditions, including uraemia, AA were determined in whole blood, which also includes AA from white blood cells and platelets. In this thesis, muscle, plasma and RBC were sampled simultaneously and reversed-phase HPLC was used to determine free AA in these compartments. RBC were separated from plasma, white blood cells and platelets, and then hemolysed, deproteinised, filtered and analysed for AA.

In study I, 27 healthy subjects were investigated in order to establish reference muscle, plasma and RBC free AA levels. These results may assist the clinical investigator when comparing the AA profiles in muscle, plasma and RBC in various disease conditions and also for evaluating the effect of various physiological stimuli on AA concentration changes in these compartments.

In study II, muscle biopsy and blood samples (plasma and RBC) were obtained from 38 haemodialysis (HD), 22 continuous peritoneal dialysis (CPD) and 10 end stage renal failure patients for determination of free AA and the results compared to data obtained from study I. Several AA abnormalities in all three compartments were observed in the uraemic patients End-stage renal failure is characterised by both disturbed protein metabolism and changes in the IGF-I/IGFBP-1 axis. Protein synthesis and degradation are regulated by a number of hormones, and hormonal regulation represents an important process to maintain coordination of nutrient flows among the various organs.

In study III, a possible association between changes in AA levels and the IGF-I/IGFBP-1 axis in end-stage renal failure was investigated in 30 HD patient who had no clinical signs of malnutrition. RBC glutamate and plasma IGFBP-1 levels were elevated in the HD patients and they were positively correlated. Since high IGFBP-1 reduces the bioavailability of IGF-I, reduced bioavailability of IGF-I, due to elevated IGFBP-1 levels, may be linked AA levels and the IGF-I/IGFBP-1 axis in end-stage renal failure was investigated in 30 HD patients who had no clinical signs of malnutrition. RBC glutamate and plasma IGFBP-1 levels were elevated in the HD patients and they were positively correlated. Since high IGFBP-1 reduces the bioavailability of IGF-I, reduced bioavailability of IGF-I, due to elevated IGFBP-1 levels, may be linked to the regulation of glutamate distribution in uraemia. Increased post-absorptive plasma glutamate levels have been linked to conditions with loss of body cell mass.

In studies IV and V investigated in a rodent model how the protein content in the diet (IV and V) and moderate renal failure (V) change the intra-and extracellular AA levels (particularly glutamine and glutamate), and whether these changes are associated with circulating IGF-I and IGFBP-1 levels (IV and V) and/or muscle ASP/DNA ratios (V). Elevated RBC glutamate levels found in rats fed a low protein diet (IV) may indicate alterations in glutamate flux and interorgan nitrogen transport. The RBC and muscle glutamate relationships to the IGF-I/IGFBP-1 axis and muscle ASP/DNA ratio support the proposal that RBC glutamate or RBC glutamine/glutamate ratio may be used as markers of catabolism, and that changes in the bioavailability of IGFs are linked to the regulation of glutamate distribution.

In summary, this thesis has demonstrated that AA determination in RBC is a simple and sensitive method for detecting AA alterations, and that RBC have an important and yet not fully clarified role in AA and protein metabolism in catabolic conditions such as uraemia and protein restriction, in both man and rat.

List of scientific papers

I. Divino Filho JC, Bergström J, Stehle P, Fürst P (1997). Simultaneous measurement of free amino acid patterns of plasma, muscle and erythrocytes in healthy human subjects. Clinical Nutrition. 16: 299-305.

II. Divino Filho JC, Barany P, Stehle P, Fürst P, Bergström J (1997). Free amino-acid levels simultaneously collected in plasma, muscle, and erythrocytes of uraemic patients. Nephrol Dial Transplant. Nov;12(11): 2339-48.
https://pubmed.ncbi.nlm.nih.gov/98056161

III. Divino Filho JC, Hazel SJ, Fürst P, Bergström J, Hall K (1998). Glutamate concentration in plasma, erythrocyte and muscle in relation to plasma levels of insulin-like growth factor (IGF)-I, IGF binding protein-1 and insulin in patients on haemodialysis. J Endocrinol. Mar;156(3): 519-27.
https://pubmed.ncbi.nlm.nih.gov/98243315

IV. Divino Filho JC, Hazel SJ, Anderstam B, Bergström J, Lewitt M, Hall K (1970). Effect of protein intake on plasma an erythrocyte free amino acids, and serum IGF-I and IGFBP-1 concentrations in rats. [Submitted]

V. Divino Filho JC, Hazel SJ, Anderstam B, Suliman ME, Bergström J, Hall K (1970). Erythrocyte glutamate as a marker of catabolism during moderate renal failure and protein restrictions in rats. [Manuscript]