Serum and tissue carnitine assay based on dialysis.

Carnitine (L-beta-hydroxy-gamma-trimethylaminobutyric acid) aids mitochondrial energy production by transferring fatty acids across the membranes for beta-oxidation. We describe here a modified enzymatic assay for free serum and tissue carnitine based on dialysis to remove interfering substances in the serum, with subsequent conversion of carnitine to the acyl derivative by carnitine acetyltransferase (EC 2.3.1.7) in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid). The method compared well with a radioenzymatic assay. The reference interval for serum is 28-70 mumol/L. Patients with advanced diabetes and those undergoing valproic acid treatment displayed lower mean values; a statistically significant number of them showed serum carnitine values below the reference interval. The method was also applied to carnitine measurement in cerebrospinal fluid and human tissues.

Microtransferrinuria and microalbuminuria. I. In the diabetic human.

We studied albumin, transferrin and total protein excretion in the urine of 110 diabetics visiting a family practice department. Of these patients 18.2% had an elevated total urinary protein above the reference range (greater than 200 mg/g creatinine). Of the remaining patients (normoproteinuria), 25.5% have elevated transferrin (greater than 0.9 mg/g creatinine) while 18.8% have elevated albumin (greater than 32 mg/g creatinine). The correlation coefficient between transferrin and albumin in urine when total urinary protein is normal was 0.77. Moderate exercise increased urinary transferrin in normal subjects 950%, while for albumin the increase was 440%. These data demonstrate the usefulness of microtransferrinuria, a potentially more sensitive indicator than microalbuminuria for diabetic nephropathy.

Microtransferrinuria and microalbuminuria. II. In the rat.

We studied albumin and transferrin excretion in the normal and diabetic rat: (1) The rat secretes small concentrations of albumin and transferrin in the urine. (2) The secretion depends on the strain and was highest in the Kyoto spontaneously hypertensive rat. (3) The secretion of these two proteins in the rat is quite dependent on age and sex. The level increases dramatically with age. The secretion is much higher in the male compared to the female. This difference is observed after puberty. The changes in transferrin relative to those in albumin are much higher. (4) In streptozotocin-induced diabetes, the concentration of albumin and transferrin expressed as milligrams per liter decreases; however, the output/24 h or per gram creatinine is increased with a greater increase in transferrin output relative to that of albumin. The similarities and differences between excretion of these two proteins in the human and the rat as well as their importance are discussed.

Plasma fibrinogen levels in type II diabetics.

Plasma fibrinogen levels measured by an immunoassay method on 170 type II diabetic patients exhibited a bimodal distribution with one small population demonstrating levels greater than those of the normal reference range. The mean plasma level of fibrinogen in the type II diabetics was higher than that of the normal population. Spearman's correlations demonstrated statistically significant positive relationships in type II diabetic patients between fibrinogen levels and fasting glucose levels, serum cholesterol, glycosylated hemoglobin and urinary albumin excretion rate. These relationships suggest that increased plasma fibrinogen may be another marker for coronary heart disease complications encountered by diabetics.

Microtransferrinuria and microalbuminuria: enhanced immunoassay.

An enhanced-sensitivity immunoassay for urinary microtransferrin and microalbumin was devised based on protein precipitation with cold trichloroacetic acid followed by dissolution of the precipitate in a small volume of phosphate buffer. Samples can be concentrated 10-fold by this method while at the same time removing many of the chromogens present in urine. Concentrated samples were assayed by immunoturbidity and radial immunodiffusion. The average recovery for urinary microtransferrin was 82 percent and for microalbumin 91 percent. The reference range for 80 normal adults for microtransferrinuria and microalbuminuria is 0 to 0.9 and 5 to 32 mg per g creatinine, respectively. The same method can be used for the assay of other proteins such as B2-microglobulin in the urine or the cerebrospinal fluid.

Erythrocyte sodium concentration and 86Rb uptake in weanling Dahl rats.

Alterations in Na, K ATPase pump activity as well as erythrocyte (RBC) intracellular sodium concentration (Nai) have been demonstrated in humans and rats with established hypertension. The contribution of hypertension itself to these changes is unclear. Accordingly, we investigated RBC ion transport and plasma ouabain-like factor (OLF) in four- to five-week old normotensive Dahl salt-sensitive (DS) and salt-resistant (DR) rats on low salt diet. Although both strains were normotensive, systolic blood pressure (SBP) of DS (123 +/- 2 mm Hg) was higher than that of DR (116 +/- 1 mm Hg). No interstrain difference was evident in RBC pump activity measured as ouabain-sensitive 86rubidium (86Rb) uptake (DS = 0.277 +/- .030 and DR = 0.271 +/- .029 mumol/10(9)RBC/h) even though RBC Nai was greater in DS than DR (14.9 +/- 2.0 v 10.7 +/- 1.0 mEq/L; P less than 0.05). Plasma OLF was higher in DS than DR (28.9 +/- 4.7 v 16.5 +/- 2.3 pmol/mL; P less than 0.05), but did not correlate with RBC pump activity in either strain. RBC Nai was directly correlated with pump activity in DS (r = 0.84, P less than 0.01) and demonstrated a trend to correlate in DR (r = 0.71, P = 0.07). RBC Nai was also directly correlated with SBP in DR (r = 0.73, P less than 0.05) and DS (r = 0.70, P = 0.05). We conclude that RBC Nai is genetically determined in Dahl rats and is elevated in normotensive DS who are at risk for hypertension development.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effect of dietary salt on renal growth in Dahl salt-sensitive and salt-resistant rats.

A high salt diet has been shown to increase renal mass of intact rats, although the mechanism by which this occurs has not been investigated. We used Dahl rats that are sensitive (DS) or resistant (DR) to the hypertensinogenic effect of salt to examine changes in renal size and composition caused by a high salt diet. Renal index, deoxyribonucleic acid (DNA), protein, water content, protein/DNA ratio, and cell number and size were measured in age-matched DR and DS on a high salt diet for 7, 14, or 28 days. The results were compared with those obtained from respective rats on a low salt diet. High salt diet elevated renal index and protein in DR and DS rats at each time point. After 7 days of a high salt diet, DNA increased in DS only. Protein/DNA ratio was progressively decreased by a high salt diet in DS and remained unchanged in DR rats. Cell number was increased 35% in DS versus only 13% in DR rats at 4 weeks. Cell size decreased 24% in DS and only 11% in DR rats. These results indicate that renal growth due to hyperplasia accompanies ingestion of a high salt diet in both DR and DS rats, but the rate of growth and the mechanism through which it occurs differ between strains. This difference may be important in delineating salt sensitivity and future development of hypertension.