Influence of training volume and acute physical exercise on the homocysteine levels in endurance-trained men: interactions with plasma folate and vitamin B12.

The interrelation between physical exercise and plasma levels of homocysteine (Hcy), vitamin B(12), and folic acid has not been examined. Therefore, we investigated the influence of extensive endurance training and acute intense exercise on plasma concentrations of total Hcy, vitamin B(12), and folic acid in 42 well-trained male triathletes. Examinations and blood sampling took place before and after a 30-day endurance training period as well as before and 1 and 24 h after a competitive exercise (sprint triathlon). Following the training period, no significant change in Hcy levels could be detected for the whole group. Subgroup analysis in quartiles of training volume revealed that - as compared with the lowest quartile (low-training group: 9.1 h training/week) - athletes in the highest training quartile (high-training group: 14.9 h training/week) exhibited a significant decrease in Hcy levels (from 12.7 +/- 2.3 to 11.7 +/- 2.4 micromol/l as compared with levels of 12.5 +/- 1.5 and 12.86 +/- 1.5 micromol/l in the low-training group; p < 0.05). The plasma folate levels were significantly higher in the high-training group at all points of examination (p < 0.05). 1 h and 24 h after competition, the Hcy concentration increased in all athletes independent of the previous training volume (24 h: 12.3 +/- 1.8 vs. 13.5 +/- 2.6 micromol/l; p < 0.001), although the increase was decisively stronger in the low-training group. 1 h after competition, the plasma folate concentration increased (7.03 +/- 2.1 vs. 8.33 +/- 2.1 ng/ml; p < 0.05) in all athletes. Multivariate analysis showed that the exercise-induced increase in the Hcy levels was dependent on baselines levels of folate and training volume, but not on the vitamin B(12) levels. In conclusion, although intense exercise acutely increased the Hcy levels, chronic endurance exercise was not associated with higher Hcy concentrations. Moreover, athletes with the highest training volume, exhibiting also the highest plasma folate levels, showed a decrease in Hcy levels following the training period as well as a much lower increase of the Hcy concentration after acute intense exercise. The combined effect of training and higher plasma folate levels to reduce Hcy should be investigated in future studies.

Pre-analytical conditions affecting the determination of the plasma homocysteine concentration.

In the past decade, moderately elevated homocysteine concentration has achieved wide-spread recognition as an independent risk factor for vascular diseases, such as stroke and peripheral vascular disease, as well as for an impaired nutritional status. In general, EDTA plasma is used for the determination of homocysteine. However, from the pre-analytical point of view it is important, that, when plasma is not separated from blood cells within 30 minutes, homocysteine levels increase in samples significantly by about 10% per hour. This 10% increase is very important, because the normal range is between 5 and 15 micromol/l and moderately elevated homocysteine concentrations above 15 micromol/l may signify an increased risk of vascular disease. These preliminary cut-off points show that there is only a small difference between normal and moderately elevated homocysteine concentrations. Most blood samples are obtained outside the hospital, and in these cases homocysteine concentrations will be falsely elevated, if no precautions are taken, such as immediate centrifugation and separation of plasma and cells. This aspect is critical both for clinical studies and in patient care outside the hospital. But even in the hospital it is difficult to separate plasma and cells within 30 minutes. In the past, different approaches were adopted to solve this problem. Potential stabilisers were sodium fluoride (4 g/l) and 3-deazaadenosine (100 micromol/l). Sodium fluoride initially increased the homocysteine concentration, which dropped below the initial values after 72 h. On the other hand, 3-deazaadenosine stabilised homocysteine concentrations for 24 h, but increased it within 72 h by roughly 10%. However, this stabiliser is restricted to HPLC technology but does not work reliably with immunoassays. Lysis of blood stabilised homocysteine, but homocysteine concentrations were systematically lower requiring totally new reference ranges. In addition, acidic citrate (0.5 mol/l) was evaluated, which seems to stabilise plasma homocysteine concentrations at ambient temperatures for several hours. However, small but systematic deviations at baseline are observed. This stabilisation procedure does not interfere with immunoassays. Because immunoassays will be the future method of choice for robust and easy to perform homocysteine measurements, because they easily allow the analyses of high sample numbers, homocysteine stabilisation in whole blood is still an important matter. It must be solved before homocysteine determinations are introduced as a general screening for vascular risk factors in non-specialist laboratories.

Assessment of vitamin B-12, folate, and vitamin B-6 status and relation to sulfur amino acid metabolism in neonates.

BACKGROUND: Total serum homocysteine (tHcy) has been used as an indicator of intracellular vitamin B-12, vitamin B-6, and folate status in adults, but data for neonates and infants are lacking. Vitamin B-12 deficiency may have fatal effects on neurologic development in infants; therefore, early diagnosis is crucial. OBJECTIVE: Our aim was to provide a reference range for tHcy in neonates and to explore the relation of tHcy to 1) serum vitamin concentrations, 2) the product of the transsulfuration pathway (cysteine), and 3) nutritional factors. DESIGN: tHcy, cysteine, folate, vitamin B-12, and vitamin B-6 were measured in 123 healthy, breast-fed neonates. The influence of nutrition (formula or human milk) on these variables was investigated in 60 infants. RESULTS: The mean (+/-SD) tHcy concentration was 7.8 +/- 3.1 micromol/L. tHcy showed a linear association with log vitamin B-12 (r = -0.64, P: < 0. 001), red blood cell folate (r = -0.33, P: < 0.001), and cysteine (r = 0.36, P: < 0.001). The strongest linear association was found between tHcy and the ratio of log cysteine to log vitamin B-12 (r = 0.71, P: < 0.0001). We found more neonates with probable tissue deficiencies of vitamin B-12 and folate on the basis of tHcy measurements than was expected from the analysis of serum vitamin concentrations alone (15.4% compared with 9.7%). Breast-fed infants had significantly lower vitamin B-12 concentrations and significantly higher serum tHcy and cysteine concentrations and ratios of log cysteine to log vitamin B-12 than did formula-fed infants (P: < 0.001). CONCLUSIONS: tHcy can be used as a functional indicator of vitamin B-12 and folate status in neonates. The ratio of cysteine to vitamin B-12 can be used as an additional index of impaired intracellular Hcy metabolism. tHcy and cysteine concentrations in infants are affected by nutritional factors.

Characterization of a new electrophoretically silent hemoglobin variant. Hb saale OR alpha 2beta 2 84(EF8)Thr --> Ala.

A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --> Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A.

Hemoglobin Rambam (beta69[E13]Gly-->Asp), a pitfall in the assessment of diabetic control: characterization by electrospray mass spectrometry and HPLC.

Hemoglobin (Hb) Rambam, or beta69[E13]Gly-->Asp, has been identified in a German woman also suffering from non-insulin-dependent diabetes mellitus and chronic obstructive pulmonary disease. This is the first observation of this Hb variant in a German family thus far. The detailed evaluation of its structure using electrospray mass spectrometry revealed new minor glycohemoglobin components and showed that the attachment of glucose to the beta NH2 terminus occurred at an almost identical rate in both wild-type and mutant beta-chains. However, the introduction of a carboxyl group at beta69 seems to increase the glycation of epsilon-amino groups of lysine residues. The glycemic state in the propositus was well reflected by the total glycohemoglobin concentrations but not by the Hb A1c values, which did not reflect hemoglobin glycation in this patient. This case demonstrates that Hb A1c cannot be used reliably in the management of diabetic patients carrying Hb variants such as Hb Rambam. Functional studies of the whole blood of the heterozygous carrier demonstrated extremely low oxygen affinity, which may have been caused by increased 2,3-diphosphoglycerate related to chronic obstructive pulmonary disease and hyperthyroidism. None of the clinical symptoms could be directly associated to Hb Rambam.

The diagnostic value of serum homocysteine concentration as a risk factor for coronary artery disease.

Hyperhomocysteinemia is now regarded as an established risk factor for coronary artery disease and is present frequently in the general population. However, the diagnostic value of this risk factor relative to others has only occasionally been investigated. We compared the diagnostic value of classic risk factors and of homocysteine in a retrospective case-control study in 191 cases with angiographically established coronary artery disease and 231 healthy controls. Life style habits were assessed by a detailed questionnaire. Laboratory parameters including lipoproteins and blood lipids, homocysteine, folate, and vitamin B12 were measured and their diagnostic value compared with each other by use of receiver-operator characteristic analysis. Comparison of the receiver-operator characteristic curves revealed that homocysteine significantly discriminated between cases and control subjects. High-density-lipoprotein cholesterol, triglycerides and non-esterified fatty acids also had an area under the curve significantly different from 0.5 (the area under the curve representing no discrimination). Homocysteine was weakly related to folate, vitamin B12, age and serum creatinine concentration. We conclude that hyperhomocysteinemia is at least as important as conventional risk factors for coronary artery disease and that receiver operator characteristic analysis of homocysteine is suitable to determine patients at the highest risk for coronary artery disease. Clinical trials testing the effect of homocysteine lowering by vitamin supplementation in the prevention of coronary artery disease are needed.

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively.

We have separated and quantified two new minor hemoglobins named HbA1d3a and HbA1d3b. The level of HbA1d3a was significantly higher in uremic than in non-uremic patients (3.00 +/- 0.50% vs. 1.28 +/- 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r = 0.80, n = 81, p < 0.002) and with plasma urea concentration (r = 0.78, n = 81, p < 0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA1d3a is an alpha-chain modified by carbamylation. The HbA1d3b level an diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 +/- 0.49 vs. 1.90 +/- 0.33). This was attributed to HbA1d3 modified by glycation. Indeed HbA1d3b correlated significantly with HbA1c (r = 0.71, p < 0.002) and with serum glucose level (r = 0.62, p < 0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averaged long-term uremia and glycemia in uremic and diabetic patients.

Hair iron content: possible marker to complement monitoring therapy of iron deficiency in patients with chronic inflammatory bowel diseases?

Measurements of the concentration of iron in hair from 10 patients with chronic inflammatory bowel diseases and from 10 healthy controls showed that the iron concentrations were significantly (P < 0.05) lower in patients before iron intake than in controls. Three weeks after beginning iron treatment, the hair iron concentrations were found to be significantly correlated (r = 0.68; P < 0.05) to reticulocyte counts. Changes in the hair iron concentrations were accompanied by similar changes in the concentrations of the markers most commonly used to diagnose and monitor iron deficiency. The results suggest that quantification of hair iron may be useful to complement evaluations of the body iron status.

Chromatographic evaluation of minor hemoglobins: clinical significance of hemoglobin A1d, comparison with hemoglobin A1c, and possible interferences.

Using an HPLC procedure, we evaluated > 10 minor hemoglobins (Hbs) in healthy adults, individuals on long-term aspirin therapy, diabetic subjects, and uremic patients. Hb A1c and Hb A1d3 were the most abundant and important minor Hb components, respectively, accounting for 4.10% +/- 0.50% and 3.46% +/- 0.43% of total Hb in 361 healthy subjects. Acetylated Hb A was a potential interferent in the measurement of Hb A1c. The amounts of both Hb A1d3 and Hb A1carb were significantly increased in uremic patients, indicating that these Hb adducts may be carbamylated. There was a significant correlation (r = 0.69, P < 0.0001) between the amounts of Hb A1d3 and plasma urea in uremic patients. Nonuremic subjects were clearly separated from uremic patients with regard to the Hb A1d3 content of their total Hb. Our data suggest that Hb A1d3 is useful for assessment of the uremic state and that the combination of Hb A1c and Hb A1d3 could be well-suited for simultaneous control of carbohydrate and urea metabolism.

Heterozygosity for the IVS-I-5 (G-->C) mutation with a G-->A change at codon 18 (Val-->Met; Hb Baden) in cis and a T-->G mutation at codon 126 (Val-->Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia.

We have analyzed the hemoglobins of a young German patient with beta-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the beta-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the GTG-->GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or alpha 2 beta (2)126(H4)Val-->Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G-->C) mutation that leads to a rather severe beta(+)-thalassemia and the GTG-->ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered beta-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G-->C) thalassemic mutation. The chromosome with the codon 18 (GTG-->ATG) and the IVS-I-5 (G-->C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G-->C) mutation living in different countries failed to detect the codon 18 (GTG-->ATG) change.

Coupling of m-aminophenylboronic acid to s-triazine-activated Sephacryl: use in the affinity chromatography of glycated hemoglobins.

An improved process is described for covalent coupling of m-aminobenzeneboric acid to s-triazine-activated Sephacryl matrices. The derivatized Sephacryl gel contained up to 150-200 mumol boronate per ml. It has been applied to the separation of glycated and non-glycated hemoglobins (Hbs) present in red-cell hemolysate. The new bioaffinity support was evaluated by the analysis of 67 diabetic patients and 20 normal adults. The mean value for glycated Hb was 6.6 +/- 0.8% for non-diabetics and 11.2 +/- 2.9% for diabetics. The method effects group-specific separation between glycated and non-glycated Hbs even in presence of foetal Hb and abnormal Hb variants. There is an excellent correlation between the glycated Hb levels obtained by the new method and two established procedures, namely high-performance liquid chromatography (r = 0.933) and affinity Merckotest (r = 0.991). The inter-assay and intra-assay coefficient of variations of less than 3.0% suggest that the method is reproducible. The results indicate that the method may serve as an alternative procedure for the study of glycated proteins. The s-triazine-activated Sephacryl could also be used for immobilizing enzymes and for preparing biospecific absorbents.

A case of the Hb Regina (beta 96 (FG3) Leu----Val) in a German male associated with high oxygen affinity and erythrocytosis.

Hb Regina was identified in a 58-year-old German male and 2 of his 3 children. All affected subjects presented moderate erythrocytosis and the whole blood exhibited increased oxygen affinity (P50:17.5 mm Hg). This hemoglobinopathy was undetectable with the conventional electrophoretic methods. It was, however, separated and quantified by cation-exchange and reverse-phase high-performance liquid chromatography. Hb Regina accounted for 30-35% of the total Hb. No significant clinical symptoms were found to be related to this hemoglobinopathy. This is the first known instance in Germany, and so far the second case reported.

High-performance liquid chromatographic separation of human haemoglobins. Simultaneous quantitation of foetal and glycated haemoglobins.

A high-performance liquid chromatographic system, which uses a weak cation exchanger (PolyCATA) together with Bis-Tris buffer (pH 6.47-7.0) and sodium acetate gradients, is described. Samples from adults and newborns were analysed and a clean separation of many minor and major normal and abnormal haemoglobin (Hb) variants was greatly improved. The method allows the separation of minor foetal haemoglobin (HbF) variants and the simultaneous quantitation of HbF and glycated HbA. HbF values correlated well with those obtained by the alkali denaturation method (r = 0.997). The glycated haemoglobin (HbAIc) levels measured in patients with high HbF concentrations correlated with the total glycated haemoglobin determined by bioaffinity chromatography (r = 0.973). The procedure is useful for diagnostic applications and affords an effective and sensitive way of examining blood samples for haemoglobin abnormalities.

High-performance liquid chromatographic separation and quantitation of glycosylated hemoglobin A2 as an alternate index of glycemic control.

By a combination of DEAE-cellulose chromatography and cation-exchange high-performance liquid chromatography glycosylated components of hemoglobin (Hb) A2 were separated and quantitated from persons with diabetes and some common hemoglobinopathies. Hb A2Ic values correlated well with total glycosylated Hb levels assayed by affinity chromatography, and Hb AIc, Hb SIc and Hb CIc levels, determined by high-performance liquid chromatography. The results indicate that Hb A2Ic may serve as an alternate index of glycemic control.

New less temperature-sensitive microchromatographic method for the separation and quantitation of glycosylated hemoglobins using a non-cyanide buffer system.

This paper describes a new microchromatographic method on Bio-Rex 70 ion-exchanger, which enables the isolation and quantitation of the minor components hemoglobin AIa+b and hemoglobin AIc. The method relies on the equilibration of the resin in a polyphosphate buffer with a pH closer to the pKa of the carboxylic group of the resin, and on the adjustment of the sample pH at 5. This induces linear pH and ionic strength gradient during the elution of the hemoglobin components. The method is little affected by temperature between 20 and 30 degrees C, by the presence of the aldimine Schiff base, and is not expected to be greatly influenced by moderate fluctuations in pH and ionic strength of the buffers used. There was good correlation between the values obtained by the new micromethod and four procedures currently used, namely high-performance liquid chromatography (r = 0.96), and Trivelli Bio-Rex 70 macromethod (r = 0.99), bioaffinity chromatography (r = 0.98), and Isolab hemoglobin AI kit (r = 0.91). The method is reproducible, the interassay and the intra-assay correlation coefficients did not exceed 4.2%. The mean value for hemoglobin AIc was 4.6 +/- 0.35% for eleven non-diabetics and 8.9 +/- 2.6% for twenty-one diabetics.

Continuous-flow analysis for glucose with use of glucose dehydrogenase immobilized in glass tubes.

We describe a method for determining glucose in serum with glucose dehydrogenase immobilized on the inner walls of glass tubes. The reactor was incorporated into a channel of a continuous-flow analyzer (the Technicon SMAC) and used daily for four weeks in routine analysis for serum glucose. During this period we tested the linearity, precision, accuracy, and durability of the reactor. Results correlated well (r = 0.9949) with those obtained by routine methods with free (nonimmobilized) glucose dehydrogenase. The method is shown to be practicable for use in the routine laboratory.

Quantitation of glycosylated hemoglobin. Elimination of labile glycohemoglobin during sample hemolysis at pH 5.

A simple method for the elimination of labile glycohemoglobin in the chromatographic quantitation of glycosylated hemoglobin is described. Use is made of the instability of Schiff base adducts in acidic solution. Erythrocytes are lysed with a pH 5 buffer. At this pH dissociation reaches completion during sample preparation.