Conversion of MM creatine kinase isoforms in human plasma by carboxypeptidase N

This study was undertaken to identify the carboxypeptidase(s) (CPase) in plasma mediating sequential conversion of the tissue isoform of the MM isoenzyme of creatine kinase (MM3 CK) to MM2 and MM1 isoforms and to elucidate relationships between CPase activity measured in plasma and observed rates of isoform conversion in vitro. Purified MM3 was incubated at 37 degrees C in plasma from normal subjects and patients with acute myocardial infarction. Isoforms were quantified by chromatofocusing. Preincubation with antiserum to CPase N prevented conversion of added MM3 to MM2 and MM1. Isoform conversion rates in the absence of antibody were proportional to plasma CPase N activity assayed spectrophotometrically by hydrolysis of furylacryloyl-L-alanyl-L-lysine substrate (r = 0.89, n = 8). Plasma CPase N activity varied by nearly 300% among individuals, but average activity was similar in samples from normal subjects (267 +/- 45 [SD] U/L, n = 18), those from outpatients with angina (289 +/- 43 U/L, n = 9), and those obtained at hospital admission from patients with acute infarction (Q wave: 279 +/- 70 U/L, n = 16; non-Q wave: 272 +/- 61 U/L, n = 14) or unstable angina (280 +/- 71 U/L, n = 11). In patients with Q wave infarction, CPase N activity increased by 43% +/- 25% between 48 hours and 72 hours (P less than 0.005 compared with admission) with a concomitant change in the rate of conversion of isoforms. Thus, the rate of conversion of isoforms in individual subjects can be estimated by assay of CPase N activity in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase of plasma nonesterified fatty acid concentration and decrease of albumin binding affinity after intravenous injection of glycocholate-lecithin mixed micelles

Lipophilic drugs intended for intravenous use can be solubilized by mixed-micellar systems containing glycoholic acid and lecithin (MM). Our present studies determined the influence of such MM preparations on albumin binding of monoacetyldiaminodiphenyl sulfone (MADDS), a deputy ligand for bilirubin. After intravenous administration of MMs to healthy male and female adult volunteers, concentration-time profiles of bile acid and nonesterified (NEFA) and esterified fatty acids were obtained as well. In vitro experiments with blood from adults and from neonatal cords indicated a modest reduction in reserve albumin for binding of MADDS after addition of MMs, resulting from glycocholic acid in the micellar preparation. After injection of MM preparations with up to 530 mg glycocholic acid, a rapid decrease of the reserve albumin was observed. The effect was more pronounced in men than in women and resulted in different areas under the time curve for the decrease (p = 0.049). At their maximum (3 to 10 minutes after MM doses) the decreases averaged (+/- SD) 68% +/- 14% in men and 45% +/- 8.5% in women. Low reserve albumin concentrations were maintained over 20 minutes despite rapidly declining bile acid concentrations. Injection of MM caused a drastic increase of NEFA in the serum samples with a more pronounced effect in men (average +/- SD increase: 473% +/- 93%) than in women (148% +/- 91%) (p = 0.01). The changes in NEFA concentrations ran reciprocal to the changes of reserve albumin for binding MADDS. In all subjects the increase in NEFA was accompanied by a decrease in reserve albumin for palmitate. Fatty acid binding to albumin was well restored within 1 hour. Thus, before drugs incorporated in MM can be prescribed to neonates who are at risk for having kernicterus, the impact of intravenous MM on bilirubin binding and NEFA levels must be investigated in that patient population.     

Simultaneous measurement of free and esterified fatty acids by gas chromatography from normal and type IV hyperlipoproteinaemic sera

A transmethylated reaction of esterified fatty acids with sodium- methoxide in a mixture of serum, petroleum ether and methanol is presented. In the conditions used the free fatty acids in the sample were not esterified. 1-2 microliters of the organic phase was injected into an OV-351 fused silica capillary column of gas liquid chromatography (GLC) fitted with flameionizing detector (FID) temperature program, and calculating integrator. By this method both free and esterified fatty acids were measured in a single run. The development of the present method for the better quantitation of free fatty acids is in progress as using gas chromatography/mass spectrometry for identification of the peaks. In thirteen healthy subjects, the serum free fatty acid content was 538 +/- 176 mumol/l and free glycerol 107 +/- 39 mumol/l, while in seven type IV hyperlipoproteinaemic sera the corresponding values were 1049 +/- 529 and 86 +/- 38 mumol/l. The most prominent differences between healthy and type IV hyperlipoproteinaemic subjects for esterified fatty acids were found in palmitic, oleic and arachidonic acids. The correlations between free and bound fatty acids has been discussed.     

Abnormal serum protein binding of acidic drugs in diabetes mellitus

Nonenzymatic glycosylation of proteins is common in diabetes mellitus and glycosylation of serum albumin in this condition has been described. To evaluate whether glycosylation of albumin affects acidic drug binding, sulfisoxazole and diazepam binding was examined in samples of normal serum incubated with glucose and in samples of serum from 42 patients with diabetes mellitus. Incubation of normal serum with 20mM glucose for several days resulted in progressive glycosylation of proteins, with decreased binding of sulfisoxazole (100 micrograms/ml) but not of diazepam (3 micrograms/ml). Free fractions of sulfisoxazole and diazepam were higher in serum from patients with diabetes. The percentage of free sulfisoxazole in serum from 10 normal subjects was 5.1% +/- 0.2%, whereas it was 16.0% +/- 1.3% in serum from 42 patients with diabetes with varying degrees of carbohydrate control. The percentage of free diazepam in plasma was 2.6% +/- 0.1% in the normal group and 3.6% +/- 0.4% in patients with diabetes. Decreased serum albumin levels, increased levels of free fatty acids, and glycosylation of plasma proteins seem to play a role in the defective acidic drug binding in diabetes. Elevated free fatty acid levels explain the abnormal binding of diazepam and the increased free fraction of sulfisoxazole is directly related to glycosylation of plasma proteins.     

S-100ao protein in serum during acute myocardial infarction

Concentrations in serum of S100ao protein (alpha alpha form of S-100 protein, which is present at high concentrations in heart muscle) were successively measured by enzyme immunoassay in 21 patients with acute myocardial infarction (AMI) and six with angina pectoris (ANP). Results were compared with measurements of creatine kinase isoenzyme MB (CK-MB) concentrations in the same specimens. Mean S100ao concentrations in sera from 100 healthy adults were 0.12 (SD 0.08) microgram/L. In patients with AMI, S100ao concentrations were 4.74 +/- 5.27 micrograms/L at admission, peaked 8 h after admission (23.5 +/- 27.7 micrograms/L), then decreased gradually. Among nine AMI patients who were admitted within an hour after their attack, eight showed abnormally high concentrations of S100ao in serum (greater than 0.5 microgram/L), whereas only four showed abnormally high CK-MB concentrations (greater than 5 micrograms/L) in sera at the time of admission. Serum S100ao concentrations remained within the normal range in all six patients with ANP; however, serum CK-MB concentrations were increased in two of them. Therefore, serum S100ao is useful not only for detection of AMI but also for differentiating AMI from ANP.     

Evaluation of hypoxic brain injury with spinal fluid enzymes, lactate, and pyruvate.

OBJECTIVE: To investigate the prognostic importance in neurologic recovery of the lumbar cerebrospinal fluid (CSF) variables creatine kinase (CK) and brain-type creatine kinase isoenzyme (CK-BB), lactate dehydrogenase (LDH) and its isoenzymes (LDH 1-5), CSF acid phosphatase, beta-D-N-acetylglucosaminidase activity, and CSF lactate, pyruvate, sodium, potassium, and calcium concentrations in patients who experienced cardiac arrest. DESIGN: Prospective clinical study with blood and CSF samples collected 4, 28, 76, and 172 hrs after resuscitation. SETTING: Medical ICU in a university hospital. PATIENTS: Twenty consecutive victims of out-of-hospital cardiac arrest. Eight patients recovered neurologically and 12 patients remained comatose or neurologically disabled until death. MEASUREMENTS AND MAIN RESULTS: CSF CK, CK-BB, LDH, and LDH isoenzyme 1-3 concentrations in all disabled patients were markedly increased at 76 hrs after the resuscitation. However, these variables were not changed in the recovered subjects. Patients (n = 7) with a mean CSF CK level of 25 +/- 33 (SD) U/L, CK-BB 23 +/- 33 U/L, and CSF lactate 3.8 +/- 0.9 mmol/L at 28 hrs after cardiac arrest remained unconscious and died. In the recovered patients, the mean CSF CK concentration was 2.0 +/- 1.5 U/L (p less than .001) and CSF lactate concentration 2.5 +/- 0.5 mmol/L (p less than .002). The lactate concentration was highest at 4 hrs after resuscitation, declining thereafter. Patients with a mean CSF total LDH level of 609 +/- 515 U/L and acid phosphatase 2.4 +/- 1.2 U/L 76 hrs after resuscitation died without regaining consciousness. In the recovered patients, the mean total CSF LDH activity was 82 +/- 58 U/L (p = .003) and CSF acid phosphatase was 0.8 +/- 0.5 U/L (p = .01) 76 hrs after resuscitation. CONCLUSIONS: CSF CK, CK-BB, and CSF lactate concentrations reflect a patient's outcome most reliably when measured within 28 to 76 hrs of the cardiac arrest. Similarly, CSF LDH, its isoenzymes 1-3, and CSF acid phosphatase concentrations, when measured at 76 hrs, can be used to monitor the patient's outcome after cardiac arrest. When correlated with Glasgow Coma Scale scores, the closest negative correlation was again seen in CSF CK and CK-BB at 28 and 76 hrs, as well as in LDH, LDH1-3, and acid phosphatase values at 76 hrs. The negative correlation between CSF lactate and Glasgow Coma Scale scores was most distinct at 28 hrs.     

Phenytoin protein binding in pediatric patients with acute traumatic injury

Phenytoin (DPH) is commonly used to treat seizures associated with acute head injury. Consequent to decreases in DPH protein binding in such patients, the DPH free fraction (DPHff) may increase and thereby produce symptoms compatible with DPH toxicity despite the presence of total serum concentrations within the usually accepted therapeutic range. We examined the effect of acute traumatic injury on DPH protein binding in 13 hospitalized pediatric patients. In addition to total and free DPH serum concentrations, biochemical variables including blood pH, total and direct bilirubin, serum urea nitrogen, creatinine, albumin, gamma glutamyltransferase (GGT), and free fatty acid concentrations were measured serially over 10 days. The DPHff was compared between selected time intervals in hospitalized patients and data obtained in a control population of 27 epileptic outpatients who were maintained on DPH. Additionally, a multiple regression model was used to examine for covariance between the DPHff and the respective biochemical variables in the hospitalized patients. In the study patients, the DPHff progressively increased, attaining a maximum value (8.5 +/- 0.7%) on the fifth hospital day which was significantly greater (6.4 +/- 0.7%, p less than .05) than that on day 1 and also in the control group (6.1 +/- 0.3%; p less than .01). Blood pH, serum albumin, free fatty acids, creatinine and bilirubin concentrations did not change, but GGT did increase significantly over the 10-day sampling period. A significant (r = .51, p less than .0001) linear relationship was found between the DPHff and the serum albumin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum carnitine concentrations in patients with idiopathic hypertrophic cardiomyopathy: relationship with impaired myocardial fatty acid metabolism.

We evaluated the clinical significance of serum carnitine concentrations in determining the severity of impaired myocardial fatty acid metabolism in idiopathic hypertrophic cardiomyopathy (HCM). We studied 56 asymptomatic or mildly symptomatic patients with HCM. Serum levels of free carnitine and acylcarnitine were measured by the enzymic cycling method. Myocardial scintigraphy with (123)I-labelled 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) was performed, and the images were analysed quantitatively and semi-quantitatively. Serum free carnitine levels were significantly higher in HCM patients than in normal subjects (52. 5+/-9.5 and 42.3+/-5.5 nmol/ml respectively; P<0.0001). On the other hand, serum acylcarnitine levels and acyl/free carnitine ratios were lower in HCM patients than in normal subjects (10.2+/-4.0 nmol/ml and 0.19+/-0.08, compared with 13.2+/-3.9 nmol/ml and 0.32+/-0.11 respectively; P<0.0001). Clinical characteristics were not significantly different between the patients showing high and normal free carnitine levels, although female patients with high free carnitine levels were few (P=0.02). Both quantitative and semi-quantitative analyses revealed that the severity of decreased myocardial BMIPP uptake was significantly correlated with serum free carnitine levels (quantitative analysis: r=-0.422, P<0.0012; semi-quantitative analysis: r=0.633, P<0.0001). In the presence of reduced carnitine uptake into the myocardium in HCM, there may also be reduced transport of acylcarnitines out of the myocardium into the plasma. Although inborn errors of fatty acid metabolism and carnitine deficiencies are reported to provoke secondary HCM and are associated with low serum carnitine concentrations, this study has revealed that the levels of carnitine are, in contrast, increased in idiopathic HCM. Moreover, serum carnitine concentrations are a sensitive indicator of the severity of impaired myocardial fatty acid metabolism even in asymptomatic patients with HCM.     

Increased lipolysis and its consequences on gluconeogenesis in non-insulin-dependent diabetes mellitus.

The present studies were undertaken to determine whether lipolysis was increased in non-insulin-dependent diabetes mellitus (NIDDM) and, if so, to assess the influence of increased glycerol availability on its conversion to glucose and its contribution to the increased gluconeogenesis found in this condition. For this purpose, we infused nine subjects with NIDDM and 16 age-, weight-matched nondiabetic volunteers with [2-3H] glucose and [U-14C] glycerol and measured their rates of glucose and glycerol appearance in plasma and their rates of glycerol incorporation into plasma glucose. The rate of glycerol appearance, an index of lipolysis, was increased 1.5-fold in NIDDM subjects (2.85 +/- 0.16 vs. 1.62 +/- 0.08 mumol/kg per min, P less than 0.001). Glycerol incorporation into plasma glucose was increased threefold in NIDDM subjects (1.13 +/- 1.10 vs. 0.36 +/- 0.02 mumol/kg per min, P less than 0.01) and accounted for twice as much of hepatic glucose output (6.0 +/- 0.5 vs. 3.0 +/- 0.2%, P less than 0.001). Moreover, the percent of glycerol turnover used for gluconeogenesis (77 +/- 6 vs. 44 +/- 2, P less than 0.001) was increased in NIDDM subjects and, for a given plasma glycerol concentration, glycerol gluconeogenesis was increased more than two-fold. The only experimental variable significantly correlated with the increased glycerol gluconeogenesis after taking glycerol availability into consideration was the plasma free fatty acid concentration (r = 0.80, P less than 0.01). We, therefore, conclude that lipolysis is increased in NIDDM and, although more glycerol is thus available, increased activity of the intrahepatic pathway for conversion of glycerol into glucose, due at least in part to increased plasma free fatty acids, is the predominant mechanism responsible for enhanced glycerol gluconeogenesis. Finally, although gluconeogenesis from glycerol in NIDDM is comparable to that of alanine and about one-fourth that of lactate is terms of overall flux into glucose, glycerol is probably the most important gluconeogenic precursor in NIDDM in terms of adding new carbons to the glucose pool.     

Free fatty acid metabolism during fenofibrate treatment of the metabolic syndrome

OBJECTIVE: Our objective was to determine whether fenofibrate modifies the metabolism of nonesterified (free) fatty acids as a component of its triglyceride-lowering action in male patients with the metabolic syndrome. DESIGN: In a placebo-controlled trial lasting 16 weeks, patients were randomly assigned to fenofibrate (200 mg/d) or placebo for 8 weeks. They were then crossed over to placebo or treatment with fenofibrate for another 8 weeks. METHODS: Thirteen adult men had clinical characteristics of the metabolic syndrome that included atherogenic dyslipidemia, hypertension, elevated fasting glucose levels, or central obesity or a combination of these. They had measurements of plasma lipid and lipoprotein levels, postheparin lipase activities, and fasting concentrations and turnover rates of nonesterified fatty acids, as well as oral glucose tolerance testing with insulin and nonesterified fatty acid measurements. Levels of apolipoprotein C-II, C-III, and B were also measured, along with levels of low-density lipoprotein cholesterol in lipoprotein species. RESULTS: Fenofibrate therapy did not change plasma concentrations and turnover rates of nonesterified fatty acids. For fasting nonesterified fatty acids, the values (mean +/- SD) for placebo versus fenofibrate were 446 +/- 31 micromol/L versus 493 +/- 71 micromol/L, respectively (not significant); nonesterified fatty acid turnover rates were 336 +/- 36 micromol/min versus 334 +/- 42 micromol/min for placebo versus fenofibrate, respectively. Moreover, no changes were noted in fasting or postprandial levels of plasma glucose and insulin. Despite this lack of change, fenofibrate therapy reduced the plasma levels of triglyceride by 30% (305 +/- 143 mg/dL versus 206 +/- 90 mg/dL for placebo versus fenofibrate, respectively; P <.045), with a similar reduction in cholesterol levels of triglyceride-rich lipoproteins. Large low-density lipoprotein species were increased and small low-density lipoprotein species were decreased by fenofibrate therapy. Levels of apolipoprotein C-III were reduced significantly (P <.03), as were ratios of postheparin hepatic lipase to lipoprotein lipase (P <.05). CONCLUSION: Fenofibrate therapy markedly reduced plasma triglyceride levels. However, it did not lower concentrations or turnover rates of nonesterified fatty acids, nor did it change glucose or insulin responses to an oral glucose challenge. These findings indicate that fenofibrate modifies fatty acid metabolism either in the liver or in triglyceride-rich lipoproteins but not in adipose tissue. Multiple mechanisms are likely involved as a consequence of the action of fenofibrate to activate peroxisomal-proliferator-activated receptor alpha.     

Effect of hypoalbuminemia and parenteral nutrition on free water excretion and electrolyte-free water resorption.

OBJECTIVE: To measure the effect of human albumin supplementation during parenteral nutrition on serum albumin concentrations, colloid oncotic pressure, free water clearance, electrolyte-free water resorption, and sodium excretion. DESIGN: Prospective, randomized, controlled trial. SETTING: Tertiary care center. PATIENTS: Thirty adult, hypoalbuminemic patients who required parenteral nutrition. INTERVENTIONS: Parenteral nutrition (control) or parenteral nutrition plus human albumin 25 g/L as a continuous infusion (treatment) for a 5-day study period. MEASUREMENTS: On days 1 and 5, serum albumin concentration, colloid oncotic pressure, free water clearance, electrolyte-free water resorption, and sodium excretion were measured. RESULTS: Serum albumin concentrations increased significantly from day 1 to day 5 in both groups (control: 2.0 +/- 0.1 [mean +/- SEM] vs. 2.3 +/- 0.1 g/dL [20 +/- 1 vs. 23 +/- 1 g/L], p = .02; treatment: 2.2 +/- 0.1 vs. 3.5 +/- 0.2 g/dL [22 +/- 1 vs. 35 +/- 2 g/L], p = .0001). Day 5 serum albumin concentrations were significantly higher in the treatment group compared with control (p = .0001). Colloid oncotic pressure increased significantly from day 1 to day 5 in the treatment group (17.8 +/- 0.8 vs. 25.1 +/- 1.0 mm Hg, p = .0001), and was significantly higher than control at day 5 (p = .0001). No significant differences were found for free water clearance, electrolyte-free water resorption, or sodium excretion within or between groups. CONCLUSIONS: In hypoalbuminemic patients, human albumin supplementation during parenteral nutrition results in significant increases in serum albumin concentrations and colloid oncotic pressure, but has no apparent effect on free water clearance, electrolyte-free water resorption, or sodium excretion.     

Association between increased concentrations of free thyroxine and unsaturated free fatty acids in non-thyroidal illnesses: role of albumin

We measured the concentrations of non-esterified free fatty acids and free and total thyroid hormones in serum from patients with various non-thyroidal illnesses (NTI) and chronic renal failure (CRF). The total concentration of free fatty acids was measured enzymatically and the eight most abundant fractions were determined by gas-liquid chromatography. The concentration of total free fatty acids was significantly increased in the NTI group as compared with controls (p less than 0.01); the concentrations of oleic, linoleic and linolenic acid were increased more than those of the other fractions. In NTI the serum-free thyroxine (FT4) concentration was increased (p less than 0.01) and the free triiodothyronine (FT3) concentration was decreased (p less than 0.001); these concentrations were measured by equilibrium dialysis. There was a significant correlation between the levels of total free fatty acids and FT4 in the NTI (n = 43) group (r = 0.45, p less than 0.01), and also between the levels of linoleic acid and FT4 (r = 0.35, p less than 0.05). The serum albumin concentration was decreased in the NTI group, and when free fatty acid to albumin molar ratios were calculated stronger correlations with FT4 were observed (total free fatty acids: r = 0.55; p less than 0.001; oleic acid: r = 0.30, p less than 0.05; linoleic acid: r = 0.46, p less than 0.01; linolenic acid: r = 0.35, p less than 0.05). There was no correlation between FT4 and unsaturated FFA concentrations in CRF patients, who had normal mean FT4 and total FFA levels. These results support the hypothesis that unsaturated fatty acids are involved in the increase of serum FT4 in NTI, especially when albumin levels are low.     

Triglyceride metabolism in acute starvation: the role of secretin and glucagon.

Plasma lipid and hormone levels have been measured during 72 hours total starvation in nine healthy subjects, to assess the relative importance of hormones and substrates in human triglyceride metabolism. Plasma free fatty acid and glycerol concentrations rose steadily on each day of starvation. Plasma triglyceride concentrations rose on the second and third days, from a control level of 649 +/- 67 mg/1 to a maximum of 1001 +/- 66 mg/1. Plasma cholesterol concentrations remained unchanged while glucose concentrations fell and insulin did not change. Plasma glucagon (C-GLI) levels doubled while secretin levels, reported previously, rose threefold. It is suggested that during acute starvation the rise in triglyceride concentration results from the increased availability of free fatty acids, and that elevated secretin and glucagon levels enhance lipolysis and hence provide substrates for triglyceride synthesis.     

Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery.

The aims of this study were to determine circulating choline status and its relationship to circulating levels of S-100beta protein and neuron-specific enolase, biochemical markers of cerebral injury and cognitive decline, after coronary artery bypass grafting (CABG) surgery. Preoperatively, patients scheduled for off-pump or on-pump CABG surgery had serum concentrations of 12.0+/-0.2 and 11.7+/-0.4 micromol/L free choline and 2640+/-65 and 2675+/-115 micromol/L phospholipid-bound choline, respectively. Serum free and bound choline levels decreased by 22-37% or 34-47% and 16-36% or 31-38% at 48 h after off-pump or on-pump surgery, respectively. Serum S-100beta and neuron-specific enolase increased from preoperative values of 0.083+/-0.009 and 6.3+/-0.2 microg/L to 0.405+/-0.022 and 11.4+/-0.8 microg/L, respectively, at 0 h postoperatively and remained elevated for 48 h after off-pump surgery. Serum free and bound choline concentrations were inversely correlated with the concentrations of S-100beta (r=-0.798; p<0.001 and r=-0.734; p<0.001) and neuron-specific enolase (r=-0.840; p<0.001 and r=-0.728; p<0.001). In conclusion, CABG surgery induces a decline in serum free and phospholipid-bound choline concentrations. The decreased serum choline concentrations were inversely correlated with the elevated levels of circulating cerebral injury markers. Thus, a decline in circulating choline may be involved in postoperative cognitive decline.     

Influence of end-stage renal failure on concentrations of free apolipoprotein A-1 in serum

We determined, immunoturbidimetrically, the concentrations of apolipoprotein A-I (apo A-I) (a) in total serum, (b) in total lipoproteins, and (c) in the fraction of d greater than 1.21 kg/L (free apo A-I) in 31 uremic patients (16 on hemodialysis, HD, and 15 with end-stage renal failure, ESRF) and 14 control subjects. The concentration of free apo A-I in serum was significantly increased in both groups of uremic patients (0.27 +/- 0.07 g/L for HD and 0.22 +/- 0.08 g/L for ESRF, mean +/- SD), in comparison with the control group (0.14 +/- 0.04 g/L). The ratio of total apo A-I to high-density-lipoprotein cholesterol was significantly increased in sera from both uremic groups, whereas the apo A-I/HDL-chol ratio of total lipoproteins was similar in all three groups. No apo A-I could be detected in five ultrafiltrates of plasma, even after 100-fold concentration. Analysis of apo A-I isoforms by isoelectrofocusing revealed a significant relative increase in apo A-I3 and a decrease in apo A-I5 isoform in ESRF patients, but not in HD patients. Finally, we found no relationship between serum TG and free apo A-I concentration.     

Effect of long-term storage on the concentration of albumin and free fatty acids in human sera.

Stored sera from healthy persons can be used to study relationships between blood variables at the time of sampling and disease appearing several years later but storage may influence the variables. In this work we measured the concentration of albumin and free fatty acids (FFA) in samples from the JANUS serum bank of Norway. Sera from blood donors and persons participating in health screening programs have been added to the bank since 1973. The concentration of albumin and FFA was measured in 443 JANUS bank sera. The material was divided into quartiles according to the length of storage: <3 years (n = 110), 3-6 years (n = 110), 6-12 years (n = 115) and >12 years (n = 108). Albumin was measured colorometrically using the bromcresol green method and FFA was determined enzymatically. The serum albumin concentrations (mean +/- SEM) in the four groups were 55.8+/-0.6, 56.2+/-0.5, 59.9+/-0.6 and 59.5+/-0.6 g/L. The values of groups 3 and 4 were significantly higher than those of groups 1 and 2 (p<0.001). The serum FFA concentrations in the four groups were 0.56+/-0.03, 0.64+/-0.03, 0.77+/-0.03 and 0.85+/-0.04 mmol/L, i.e. a significant storage effect. The Scheffé multiple comparison test showed that FFA values in groups 3 and 4 were significantly higher than those in groups 1 and 2 (p <0.001 for group 4 vs. 1 and 2, and 3 vs. 1; p<0.04 for group 3 vs. 2) Serum FFA and albumin levels were positively associated (r = 0.489, p <0.01). Using linear regression analysis, it was estimated that serum albumin values increased by 0.28 g/L per year (i.e. 0.5%) and FFA by 0.02 mmol/L (i.e. 3.8%). Thus, measured by standard methods, serum FFA and albumin could increase in response to several years of storage at -25 degrees C. It is suggested that the storage time dependent increase in FFA is due to FFA liberation from lipoprotein triglycerides, whereas the apparent increase in albumin concentration possibly could be attributed to an unfolding of the protein, allowing more bromcresol green to be bound.     

Immunonephelometric determination of retinol-binding protein in serum and urine

An immunonephelometric method developed for measurement of retinol-binding protein (RBP) in serum and urine can detect it in concentrations of about 30 micrograms/L, which is in the lower limit of its normal concentration in urine (range 0-0.56 mg/L; mean +/- SD 0.19 +/- 0.15; n = 44). Urinary RBP was increased (range 0.93-29.5 mg/L) in all of 25 urine specimens from 13 subjects being treated with aminoglycoside (tobramycin). Urinary excretion of RBP was correlated (r = 0.83) with the excretion of beta 2-microglobulin. The within-assay and day-to-day precision (CV) was determined over the detection range of 0.03-8 mg/L. Within these limits the corresponding CVs varied from 4 to 27% and from 8 to 30%, respectively. The method had fairly good precision within the optimal measuring range of approximately 0.4 to 4.5 mg/L for both urine and 20-fold diluted serum samples. For various RBP concentrations our analytical recovery was 89-114% of added RBP. Results by this method correlated well (r = 0.96, n = 24) with those by a radial immunodiffusion method.     

Enzymic determination of free fatty acids in serum

We describe the enzymic determination of free fatty acids in serum with use of acyl-CoA synthetase (EC l.2.1.3), acyl-CoA oxidase (no EC no. assigned) and peroxidase (EC 1.11.1.7). The free fatty acids are activated by acyl-CoA synthetase in the presence of ATP and coenzyme A. The acyl-CoA formed is oxidized by acyl-CoA oxidase to enoyl-CoA with simultaneous production of hydrogen peroxide, which is oxidatively coupled with 4-amino-antipyrine and 2,4-dibromphenol in the presence of peroxidase to yield a product that absorbs maximally at 505 nm. Standard curves prepared with various kinds of fatty acids are linear to at least 2.0 mmol/L and are practically congruent. Ascorbic acid or bilirubin interferes slightly. Results by the present method correlate well with those by the colorimetric method involving 2-(2-thiozolylazo)-p-cresol (r = 0.98). Replicate analyses of standard solution and of two kinds of control sera demonstrated the following between-assay precision: mean absorbance 0.224 (SD 0.002), CV 0.85%; mean concentration, 326 (SD 7.3) and 1076 (SD 22.7) mumol/L, CV 2.24 and 2.11%, respectively.     

New ultrafiltration method for free thyroxin compared with equilibrium dialysis in patients with thyroid dysfunction and nonthyroidal illness

This new ultrafiltration method for free thyroxin in serum [FT4(U)] is based on radioimmunoassay of the free hormone fraction in ultrafiltrates obtained by centrifuging serum samples in Unisep Ultracent-10 ultrafiltration devices. We compared the results obtained with those by an equilibrium dialysis method [FT4(D)]. In 36 euthyroid healthy subjects, the mean FT4(U) concentration was 24.2 pmol/L and the mean FT4(D) concentration 14.8 pmol/L. In hyperthyroid and hypothyroid patients, results by the ultrafiltration method were also approximately twice as high as those obtained by the dialysis method. In 23 patients with various nonthyroidal illnesses, mean FT4(U) was 41.2 pmol/L and mean FT4(D) 19.8 pmol/L. The mean FT4(U)/FT4(D) ratio in patients with nonthyroidal illnesses (1.97) was not significantly higher than in control subjects (1.68), making it unlikely that the increase in serum FT4 is caused by weakly protein-bound and therefore dialyzable inhibitors of thyroxin binding to carrier proteins. However, two nonthyroidally ill patients with a clearly increased FT4(U) but a normal FT4(D) concentration might have had such inhibitors, whereas for two other nonthyroidally ill patients a high molar ratio of free fatty acids to albumin is a more likely explanation for increased FT4(U) and FT4(D) concentrations. On theoretical grounds, we consider the FT4(U) concentrations analytically more nearly accurate than FT4(D) values for all patient groups studied.